May 2011
Volume 52, Issue 6
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Immunology and Microbiology  |   May 2011
Engagement of TLR2 Reverses the Suppressor Function of Conjunctiva CD4+CD25+ Regulatory T Cells and Promotes Herpes Simplex Virus Epitope-Specific CD4+CD25 Effector T Cell Responses
Author Affiliations & Notes
  • Gargi Dasgupta
    From the Laboratory of Cellular and Molecular Immunology, Gavin Herbert Eye Institute, School of Medicine, and
  • Aziz Alami Chentoufi
    From the Laboratory of Cellular and Molecular Immunology, Gavin Herbert Eye Institute, School of Medicine, and
  • Sylvaine You
    INSERM U580, Hôpital Necker-Enfants Malades, Paris, France.
  • Payam Falatoonzadeh
    From the Laboratory of Cellular and Molecular Immunology, Gavin Herbert Eye Institute, School of Medicine, and
  • Lourie Ann A. Urbano
    From the Laboratory of Cellular and Molecular Immunology, Gavin Herbert Eye Institute, School of Medicine, and
  • Ayesha Akhtarmalik
    From the Laboratory of Cellular and Molecular Immunology, Gavin Herbert Eye Institute, School of Medicine, and
  • Kimberly Nguyen
    From the Laboratory of Cellular and Molecular Immunology, Gavin Herbert Eye Institute, School of Medicine, and
  • Lilit Ablabutyan
    From the Laboratory of Cellular and Molecular Immunology, Gavin Herbert Eye Institute, School of Medicine, and
  • Anthony B. Nesburn
    From the Laboratory of Cellular and Molecular Immunology, Gavin Herbert Eye Institute, School of Medicine, and
  • Lbachir BenMohamed
    From the Laboratory of Cellular and Molecular Immunology, Gavin Herbert Eye Institute, School of Medicine, and
    the Center for Immunology, University of California at Irvine, Irvine, California; and
  • Corresponding author: Lbachir BenMohamed, Laboratory of Cellular and Molecular Immunology, University of California Irvine Medical Center, 101 The City Drive, Building 55, Room 202, Orange, CA 92868; lbenmoha@uci.edu
Investigative Ophthalmology & Visual Science May 2011, Vol.52, 3321-3333. doi:10.1167/iovs.10-6522
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      Gargi Dasgupta, Aziz Alami Chentoufi, Sylvaine You, Payam Falatoonzadeh, Lourie Ann A. Urbano, Ayesha Akhtarmalik, Kimberly Nguyen, Lilit Ablabutyan, Anthony B. Nesburn, Lbachir BenMohamed; Engagement of TLR2 Reverses the Suppressor Function of Conjunctiva CD4+CD25+ Regulatory T Cells and Promotes Herpes Simplex Virus Epitope-Specific CD4+CD25 Effector T Cell Responses. Invest. Ophthalmol. Vis. Sci. 2011;52(6):3321-3333. doi: 10.1167/iovs.10-6522.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: The authors recently reported that Foxp3+CD4+ CD25+(Bright) “natural” regulatory T cells (nTreg cells) are abundant in rabbit conjunctiva and suppress herpes simplex virus (HSV)-1–specific CD4+ and CD8+ effector T cells (Teff cells). However, little is known about the overall regulatory mechanisms of these nTreg cells. The authors investigate the regulation of conjunctiva-resident nTreg cells through Toll-like receptors (TLRs) and their effect on ocular mucosal Teff cell immunity.

Methods.: CD4+CD25+ nTreg cells were purified from naive rabbit conjunctivas, and their TLR expression profile was determined. The effects of TLR engagement on nTreg cell-mediated suppression of CD4+ Teff cells were determined in vitro and in vivo.

Results.: The authors found that conjunctiva-resident nTreg cells express high levels of TLR2 and TLR9; exposure to the TLR2 ligand lipoteichoic acid (LTA) led to the increased activation and proliferation of nTreg cells, and the addition of autologous APCs further increased nTreg cell expansion; in contrast, the TLR9 ligand CpG2007 inhibited the proliferation of nTreg cells, and the addition of autologous APCs had no effect on such inhibition; nTreg cells treated with LTA, but not with CpG2007, expressed IFN-γ and IL-10 mRNA, but not TGF-β; consistent with in vitro data, rabbits immunized by topical ocular drops of HSV-gD peptides + TLR2 ligand (LTA) displayed enhanced CD4+CD25 Teff cell immune responses when compared with HSV-gD peptides + TLR9 ligand (CpG2007).

Conclusions.: Although conjunctiva-resident CD4+CD25+ nTreg cells express high level of TLR2 and TLR9, their suppressive function is more significantly reversed after the administration of TLR2 ligand (LTA; P < 0.005) than of TLR9 ligand (CpG200; P > 0.005). These findings will likely help optimize the topical ocular administration of immunotherapies.

Regulatory T cells (Treg) constitute a phenotypically and functionally distinct αβ T cell population that represents 1% to 2% of peripheral mononuclear cells and 5% to 10% of all peripheral CD4+ T cells in both mice and humans. 1,2 Naturally occurring CD4+CD25+ regulatory T cells (also known as nTreg) have emerged as a dominant regulatory T cell subpopulation mediating peripheral adaptive immune tolerance and suppression of immunity against many pathogens. 2 7 In general, most nTreg cells constitutively express high-affinity IL-2Rα receptor (i.e., CD25) and the transcription factor forkhead/winged-helix transcription factor (Foxp3) molecules. 8,9 In addition, in the resting state, they constitutively express several activation markers, such as glucocorticoid-induced receptor TNF-R (GITR), OX40 (CD134), and CTLA-4. 10,11 The mucosal epithelia and underlying lamina propria contain large numbers of regulatory T cells that play an important role in maintaining mucosal homeostasis and defense against external pathogens. 12  
We recently described an abundance of “natural” Foxp3+ CD4+CD25+(Bright) nTreg cells in “nonimmune” rabbit conjunctiva, the main inductive site of the ocular mucosal immune system. 1 We demonstrated that conjunctiva-resident nTreg cells suppress herpes simplex virus type 1 (HSV-1)-specific CD4+ and CD8+ effector T cells (Teff). Converging evidence, from our laboratory and other laboratories, demonstrates that nTreg cells have the potential to dampen the vaccine-induced, HSV-specific, Teff-mediated immunity. 13 16 Despite recent extensive studies on nTreg cells, the molecular mechanism by which nTreg cells mediate the suppression of pathogen-specific T cell immunity or dampen vaccine-induced Teff cells remains poorly understood. 
Recent discovery of Toll-like receptors (TLRs) has provided the link between the innate and adaptive immune systems. 17 19 At least 13 TLRs that recognize a limited but conserved set of ligands from viral, bacterial, protozoan, and helminth pathogens have now been identified in mice and humans. 20 23 It was initially thought that TLRs are primarily expressed by antigen-presenting cells (APCs) such as macrophages and dendritic cells and that interactions between microbial ligands and TLRs in these cells would indirectly result in the activation of effector T cells (Teff cells). However, it has become evident that TLRs are also expressed by conventional αβ T cells, regulatory T cells, and γδT cells and by natural killer T cells. 21,24 26 A correlation between nTreg cell–suppressive function and TLR expression has been documented. 27 29 Although some TLR ligand-receptor interactions have been proposed to increase nTreg cell suppressive capacity, others have been shown to limit such function. 28,30 32 For example, TLR2 signaling temporally abrogates the suppressive phenotype of nTreg cells and decreases Foxp3 expression. 33,34 A direct involvement of TLR-5 on nTreg cell immunosuppressive function has been demonstrated recently. 28 TLR9 ligand CpG DNA also modulates adaptive immune responses by inhibiting the suppressive effects of nTreg cells in mice. 32 However, the phenotypic and functional characterization of TLRs, particularly on conjunctiva-resident nTreg cells, has not been reported. 
In the present study, we examined the expression profile of TLRs on conjunctiva-resident nTreg cells and assessed whether TLRs are functionally active by assessing whether stimulation of these nTreg cells with TLR agonists, in the absence of APCs, would result in modification of their regulatory functions. We show that rabbit conjunctiva-resident CD4+CD25+ nTreg cells express high levels of functional TLR2 and TLR9. Topical ocular immunization of rabbits with HSV-gD peptide T cell epitopes, together with a TLR2 ligand (LTA), reverses CD4+ CD25+ nTreg cell suppressive function. In contrast, topical ocular immunization of rabbits with the same epitopes delivered with a TLR9 ligand (CpG2007) resulted in only a slight effect on CD4+CD25+ nTreg cell suppressive function. Our findings demonstrate that regulating conjunctiva CD4+CD25+ nTreg cell function trough TLR2 and TLR9 is possible and leads to the modulation of ocular mucosal HSV-specific CD4+CD25 Teff cell responses. The potential use of TLR agonists for steering ocular mucosal T cell responses in a topical ocular therapeutic vaccine strategy is discussed. 
Materials and Methods
Rabbits
For in vitro studies, healthy rabbit eyes with palpebral conjunctiva were purchased from Pel Freeze Biologicals (Rogers, AR). For in vivo studies, 7-week-old New Zealand White female rabbits (Harlan, Indianapolis, IN) were used for all experiments. Rabbits were maintained under specific pathogen-free conditions in the animal facilities of the University of California at Irvine. 
Media, Reagents, and Antibodies
Cells were cultured in RPMI-1640 supplemented with 10% heat-inactivated FCS, 100 IU/mL penicillin, 100 μg/mL streptomycin, 2 mM l-glutamine, and 1 mM Na-pyruvate at 37°C, 5% CO2 incubator. Lipoteichoic acid (LTA) and phytohemagglutinin were purchased from Sigma. CpG2007 was purchased from Coley Pharmaceutical Group (Wellesley, MA). Anti-human TLR2-PE, TLR3-PE, TLR4-PE, TLR8-PE, and TLR9-PE monoclonal antibodies were purchased from Imgenex (San Diego, CA). Purified anti-human CD3 was purchased from R&D Systems (Minneapolis, MN). CFSE was purchased from Invitrogen (Carlsbad, CA). Anti-rabbit CD4, anti-rabbit CD25, and goat anti-mouse IgG-PE were purchased from Antigenix America (Melville, NY). Rabbit anti-mouse IgG-PE was purchased from Serotec (Raleigh, NC). Monoclonal anti-rabbit CD11b-PE, CD11b-FITC, CD11c-PE, and CD11C-FITC were purchased from Chemicon (Temecula, CA). Pam3CSK4 was purchased from Imgenex (catalog no. IMG-2201). PE-conjugated anti-human Foxp3 was purchased from e-Bioscience (San Diego, CA; catalog no. 12–4776-71). Purified anti-human Foxp3 was purchased from BioLegend (San Diego, CA; catalog no. 320101). 
Purification of CD4+CD25+ and CD4+CD25 Cells
Single cell suspension from healthy rabbit conjunctiva was prepared as previously described. 1 CD4+CD25+ nTreg cells were purified from conjunctiva-derived single cell suspension by positive selection with magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). In brief, 18 to 20 × 107 single cell suspension in 2 mL MACS buffer (Miltenyi Biotec) was first incubated with 100 to 120 μL anti-rabbit CD25-biotin followed by a second incubation with 360 μL anti-biotin microbeads (Miltenyi Biotec) according to manufacturer's instructions. CD25+ cells were subsequently eluted from the MACS column. The flow-through containing 9 to 10 × 107 CD25+ cells was collected and further purified by two successive incubations, the first with 100 μL anti-rabbit CD4-FITC and the second with 100 μL anti-FITC microbeads (Miltenyi Biotec) according to manufacturer's instructions. CD4+CD25+ cells were subsequently eluted from the MACS column. The flow-through containing CD4CD25 cells was collected and used as a source of APCs. 
Purification of CD11b+ Cells
Single cell suspension from healthy rabbit conjunctiva was prepared as described earlier. 1 Cells were plated in a T75 flask at a density of 10 × 106/20 mL culture media and were allowed to attach for 2 hours at 37°C in a 5% CO2 incubator. Nonattached cells were removed by aspiration. Attached cells were gently washed two times with culture media, scraped, and collected in culture media. Trypan blue staining was used to check cell viability. The expression of CD11b+/CD11c+ cells in the attached cells was checked by staining with anti-rabbit CD11b-FITC or anti-rabbit CD11c-PE antibody. The purity of CD11b+ cells was 95% as checked by flow cytometry (data not shown). The average yield of CD11b+/CD11c+ cells from 30 healthy conjunctivas was 20% (data not shown). 
Intracellular Staining of TLRs
Purified CD4+CD25+ cells (0.24 × 106 cells/100 μL culture medium) were washed and suspended in FACS staining buffer (PBS [pH 7.4] containing 0.5% BSA, 0.02% sodium azide). Cells were stained with 1 μL anti-rabbit CD25-biotin for 30 minutes at room temperature, washed, and further incubated with 1 μL anti-strepatavidin-Cy5 (Chemicon) for 30 minutes at 4°C in the dark. Cells were washed twice with FACS staining buffer. For intracellular staining, cells were fixed with fixation-permeabilization buffer (e-bioscience) according to the manufacturer's instruction. Cells were then stained with 3 μL anti-human TLR2-PE, TLR3-PE, TLR4-PE, TLR8-PE, and TLR9-PE for 30 minutes at 4°C in the dark. Cells were washed twice with FACS staining buffer and analyzed by flow cytometry (FACScan; BD Biosciences, San Jose, CA). The acquired data were analyzed by acquisition and analysis software (Cell Quest; BD Biosciences). 
Intracellular Staining of Foxp3
Harvested CD4+CD25+ cells (0.5 × 106 cells/assay) from a 96-well tissue culture plate were washed and suspended in FACS staining buffer (PBS [pH 7.4] containing 0.5% BSA, 0.02% sodium azide). Cells were surface stained with anti-rabbit CD4-FITC (10 μL/assay) for 30 minutes at 4°C in the dark. For intracellular staining, cells were fixed as described and were stained with anti-human foxp3-PE (20 μL/assay) for 30 minutes at 4°C in the dark. The rest of the procedure was same as described. 
CFSE Labeling of CD4+CD25+ Cells
Purified CD4+CD25+ or CD4+CD25 cells at a density of 1 × 106 cells/mL PBS were labeled with a final concentration of 2.5 μM CFSE for 10 minutes at 37°C according to the product instructions. The reaction was quenched by the addition of 5 vol culture media. Cells were washed three times with culture media before they were placed in culture. 
Proliferation CD4+CD25+ T Cells
Freshly isolated and CFSE-labeled CD4+CD25+ nTreg cells (5 × 104 cells/well in 200 μL) were plated in a 96-well, flat-bottomed tissue culture plate in culture media. Cells were stimulated with either soluble anti-human CD3 (1 μg/mL) alone or with a combination of soluble anti-CD3 (1 μg/mL) or autologous APCs (1 × 105 APCs/well in 200 μL) for 6 days at 37°C in a 5% CO2 incubator. To test the effect of TLR ligands on the proliferation of CD4+CD25+ nTreg cells, in a second set, CFSE-labeled CD4+CD25+ nTreg cells (5 × 104 cells/well in 200 μL) were incubated with either 10 μg/mL LTA (ligand for TLR2) or 1 μg/mL CpG2007 (ligand for TLR9) along with soluble anti-human CD3 (1 μg/mL) only or with a combination of soluble anti-CD3 (1 μg/mL) and autologous APCs (1 × 105 APCs/well in 200 μL). Autologous APCs were treated with 1 μg/mL mitomycin C for 45 minutes at 37°C and were washed five times before they were put into the culture. Cells were plated in triplicate and incubated for 6 days at 37°C in a 5% CO2 incubator. Cells were harvested, washed twice with FACS staining buffer, stained with anti-rabbit CD4-PE or anti-rabbit CD25-PE, and analyzed with flow cytometry. The absolute number of proliferating cells was calculated using the formula (No. events in CD4+ proliferating cells) × (No. events in gated cells)/(No. total events acquired). 
Suppression of CD4+CD25 Teff Cell Proliferation by CD4+CD25+ nTreg Cells
To monitor the activation and proliferation of Teff cells, freshly purified and CFSE-labeled CD4+CD25 effector T cells (5 × 104 cells/well in 200 μL) were stimulated with soluble anti-human CD3 antibody (1 μg/mL) and autologous APCs (1 × 105 APCs/well in 200 μL). To monitor the suppressed proliferation of Teff cells influenced by nTreg cells, in a second set, nonlabeled CD4+CD25+ nTreg cells (10 × 104/well in 200 μL) were added together with CFSE-labeled CD4+CD25 effector cells (5 × 104 cells/well in 200 μL) and stimulated with soluble anti-human CD3 antibody (1 μg/mL) and autologous APCs (1 × 105 APCs/well in 200 μL). To monitor the effect of TLR ligands on this suppression assay, in a third set, nonlabeled CD4+CD25+ nTreg cells (10 × 104/well in 200 μL) were added together with CFSE-labeled CD4+CD25 effector cells (5 × 104 cells/well in 200 μL) along with either 10 μg/mL LTA (ligand for TLR2) or 1 μg/mL CpG2007 (ligand for TLR9) and were stimulated as described with soluble anti-human CD3 antibody (1 μg/mL) and autologous APCs (1 × 105 APCs/well in 200 μL). Autologous APCs were always treated with 1 μg/mL mitomycin C for 45 minutes at 37°C and were washed five times before they were put into the culture. Cells in all three sets were plated in triplicate and incubated for 6 days at 37°C in a 5% CO2 incubator. Cells were harvested, washed twice with FACS staining buffer, and stained with anti-rabbit CD4-PE or anti-rabbit CD25-PE and analyzed by flow cytometry. The absolute number of proliferating cells was calculated using the formula (No. events in CD4+ proliferating cells) × (No. events in gated cells)/(No. total events acquired). 
Ex Vivo Assay
Six New Zealand White female rabbits (two per group) were immunized ocularly (both eyes) with a mixture of four HSV-gD peptides (gD146–179, gD287–317, gD49–82, and gD200–234) along with either TLR ligand CpG2007 (group 1) or LTA (group 2), or not immunized (group 3), at an interval of 14 days. The amount of each HSV-gD peptide in the peptide mixture was 25 μg and was suspended in 100 μL sterile PBS. The amount of CpG2007 or LTA in the peptide mixture was 25 μg/100 μL peptide mixture. Each rabbit was immunized with 100 μL (50 μL/eye) peptide mixture. Ten days after the third immunization, the conjunctiva was removed from each rabbit eye, and a single cell suspension was prepared as described earlier. 1 CFSE-labeled, conjunctiva-derived, single cell suspensions were plated at a density of 3 × 105 cells in 200 μL in a 96-well, round-bottomed tissue culture plate, stimulated with individual HSV-gD peptide (10 μg/mL) or nonspecific positive control phytohemagglutinin (10 μg/mL) and were incubated for 5 days at 37°C in a CO2 incubator. Cells were harvested and stained with anti-rabbit CD4-PE and analyzed by flow cytometry. 
Cytokine Expression Quantification Using Real-Time PCR
Purified nTreg cell lysate was suspended directly in RLT buffer (Qiagen, Valencia, CA). This lysate was then processed over RNeasy columns according to the manufacturer's instructions (Qiagen). The RNA was eluted from the silica column with 50 μL DEPC-treated water. One microliter of the product was analyzed (2100 Bioanalyzer; Agilent, Wilmington, DE) using the nano-RNA protocol to verify both the quantity and the quality of the RNA. RNA obtained from purified nTreg cells was converted to cDNA using a synthesis protocol (Sensiscript; Qiagen). For these, 1000 ng RNA was reverse transcribed in a 20-μL reaction volume after wiping out the genomic DNA. Reactions were carried out for 30 minutes at 42°C, followed by 5 minutes at 95°C and cooling to 4°C. After synthesis, the cDNA from all sources was diluted to equivalent input RNA levels with water (10 ng input RNA/μL) and was stored at −20°C until use. cDNA samples were subjected to PCR using specific primers. Primers were designed with a software program (Primer 3 Internet; The Whitehead Institute, Cambridge, MA), 35 and their specificities were confirmed by a BLAST Internet software-assisted search of the nonredundant nucleotide sequence database (National Library of Medicine, Bethesda, MD). The following primers were used for cytokine detection: IFN-γ: sense, 5′-CATCAGATGTGGCAAATGGT-3′; forward, 5′-ATCCACCGAAATTCGAGTCA-3′; IL-10: sense, 5′-AAAAGCTAAAAGCCCCAGGA-3′; forward, 5′-ATAGGATCGGGAGCTGAGGT-3′; TGF-β: sense, 5′-TGCTTCAGCTCCACAGAGAA-3′; forward; 5CTTGCTGTACTGGGTGTCCA-3′. 
All PCR reactions were carried out with 5 ng reverse-transcribed RNA, 200 nM each forward and reverse primer, and master mix (SYBR Green; Applied Biosystems, Foster City, CA) in a final volume of 25 μL. Cycling parameters were 50°C for 2 minutes and 95°C for 5 minutes, followed by 45 cycles of 95°C for 30 seconds and 60°C for 90 seconds. Plates were read after each cycle, and a melting curve was generated after amplification. For quantitative real-time PCR, samples were normalized by GADPH amplification and were amplified in triplicate using a thermal cycler (Opticon; MJ Research, Waltham, MA). PCR controls without reverse transcriptase (water control) or with normal human genomic DNA as a template were routinely negative. Cytokine expression were carried out using a 2-ΔΔCT method, as recently described. 1  
Statistical Analysis
All analyses for statistically significant differences were performed with the Student's paired t-test. P < 0.05 was considered significant. All cultures were performed in triplicate, and error bars represented standard deviation. 
Results
Conjunctiva-Resident CD4+CD25+ T Cells Express High Levels of TLR2 and TLR9
CD4+CD25+ T cells were purified from normal rabbit conjunctiva by magnetic cell separation, as we described previously. 1 The expression of TLR2, TLR3, TLR4, TLR8, and TLR9 by conjunctiva-resident CD4+CD25+ T cells was determined by intracellular staining and analyzed by flow cytometry. As shown in Figure 1A, conjunctiva-derived CD4+CD25+ T cells expressed detectable amounts of all tested TLRs. Further analysis of mean fluorescence intensity (MFI) of each TLR revealed significantly higher expression levels of TLR2 and TLR9 compared with TLR3, TLR4, and TLR8 (P < 0.005; Fig. 1C). Given that no anti-rabbit TLR mAbs were commercially available, we used a mouse anti-human TLR IgG1 mAb to detect the TLR expression profile in rabbits. The specificity of anti-human TLR mAbs to detect TLR in rabbit CD4+CD25+ T cells was verified by an isotype mAb control. Figure 1B shows the binding of mouse IgG1-PE (isotype control) to CD4+CD25+ T cells under identical experimental conditions and overlaid on the binding of anti-human TLR2 to CD4+CD25+ T cells. No binding between mouse IgG1-PE and CD4+CD25+ T cells was observed, which ruled out the possibility of any nonspecific binding between anti-human TLR-PE and CD4+CD25+ T cells. 
Figure 1.
 
Intracellular staining of Toll-like receptors in CD4+CD25+ regulatory T cells purified from rabbit conjunctiva. (A) Purified CD4+CD25+ cells (0.66 × 105 per assay) were first surface stained with 1 μL α rabbit CD25-biotin and then intra cellularly stained with 1 μL α human TLR2-PE, TLR3 PE, TLR4-PE, TLR8-PE, TLR9-PE, as shown on each histogram. Dotted line: CD4+CD25+ cells only; solid line: cells stained with anti-human TLR-PE antibody. (B) Isotype control (dotted line) where CD4+CD25+ cells (0.66 × 105) were first stained with 1 μL anti-rabbit CD25-biotin followed by staining with 1 μL mouse IgG1 PE. (C, D) Level of TLRs in CD4+CD25+ and CD4+CD25 cells as a function of MFI. *P < 0.05 when compared with the MFI of TLR2 and TLR9 expression in CD4+CD25+ and CD4+CD25 populations.
Figure 1.
 
Intracellular staining of Toll-like receptors in CD4+CD25+ regulatory T cells purified from rabbit conjunctiva. (A) Purified CD4+CD25+ cells (0.66 × 105 per assay) were first surface stained with 1 μL α rabbit CD25-biotin and then intra cellularly stained with 1 μL α human TLR2-PE, TLR3 PE, TLR4-PE, TLR8-PE, TLR9-PE, as shown on each histogram. Dotted line: CD4+CD25+ cells only; solid line: cells stained with anti-human TLR-PE antibody. (B) Isotype control (dotted line) where CD4+CD25+ cells (0.66 × 105) were first stained with 1 μL anti-rabbit CD25-biotin followed by staining with 1 μL mouse IgG1 PE. (C, D) Level of TLRs in CD4+CD25+ and CD4+CD25 cells as a function of MFI. *P < 0.05 when compared with the MFI of TLR2 and TLR9 expression in CD4+CD25+ and CD4+CD25 populations.
We also checked the TLR expression profile on CD4+ CD25 Teff cells, which was copurified along with the magnetic separation of CD4+CD25+ nTreg cells from rabbit conjunctiva. Figure 1D showed a significantly lower expression of TLR2 (MFI <50) in CD4+CD25 Teff cells compared with the TLR2 expression in CD4+CD25+ nTreg cells (MFI >250) shown in Figure 1C. Interestingly, no significant differences in the expression level of TLR9 between these two cell populations (i.e., CD4+CD25+ nTreg cells and CD4+CD25 Teff cells) were observed (Figs. 1C, 1D). This indicates the integrity of the CD4+CD25+ Treg cell and CD4+CD25 Teff cell populations. 
We next investigated whether the intracellular expression of TLR2 and TLR9 was comparable to that of APCs, an immune cell population known for high expression of TLRs. We tested the expression levels of TLRs in CD11b/c+ APCs isolated from healthy rabbit conjunctiva. Figure 2A shows the histogram representation of all tested TLRs in autologous CD11b/c+ APCs, and Figure 2B shows their corresponding MFI. Autologous CD11b/c+ APCs also expressed all tested TLRs, and the highest expression was observed for TLR9 (MFI ≥500). However, no significant difference in the expression level of TLR2 was observed in CD11b/c+ APCs or CD4+CD25+ nTreg cells (Figs. 1B, 2B), indicating a similar TLR expression profile between nTreg cells and APCs in the conjunctiva compartment. 
Figure 2.
 
(A) Intracellular staining of Toll-like receptors in CD11b+ APCs isolated from rabbit conjunctiva. CD11b+ cells (0.24 × 106 per assay) were first surface stained with 1 μL anti-rabbit CD11b-FITC antibody and then intracellularly stained with 3 μL anti-human TLR2-PE, TLR3-PE, TLR4-PE, TLR8-PE, TLR9-PE in each set. Dotted line: CD11b+ cells only; solid line: cells stained with anti-human TLR-PE antibody. (B) TLR level in CD11b+ cells as a function of MFI. *P < 0.05 compared with the MFI of TLR2 and TLR9 expression in the CD11b+ populations.
Figure 2.
 
(A) Intracellular staining of Toll-like receptors in CD11b+ APCs isolated from rabbit conjunctiva. CD11b+ cells (0.24 × 106 per assay) were first surface stained with 1 μL anti-rabbit CD11b-FITC antibody and then intracellularly stained with 3 μL anti-human TLR2-PE, TLR3-PE, TLR4-PE, TLR8-PE, TLR9-PE in each set. Dotted line: CD11b+ cells only; solid line: cells stained with anti-human TLR-PE antibody. (B) TLR level in CD11b+ cells as a function of MFI. *P < 0.05 compared with the MFI of TLR2 and TLR9 expression in the CD11b+ populations.
LTA (a TLR2 ligand) Induces the Proliferation of CD4+CD25+ nTreg Cells
To determine whether TLR2 and TLR9 receptors expressed on CD4+CD25+ nTreg cells were functional, we performed an in vitro proliferation assay for nTreg cells in the presence and absence of their specific ligands: LTA and CpG2007 DNA, TLR2 and TLR9 ligands, respectively. CFSE-labeled CD4+CD25+ nTreg cells were stimulated with either soluble anti-human CD3 or a combination of soluble anti-human CD3 and autologous APCs and were incubated for 6 days in the presence or absence of either LTA or CpG2007. The proliferation of CFSE-labeled CD4+CD25+ nTreg cells was then analyzed by flow cytometry on gated CD4+ T cells. Figure 3A shows that the proliferation of CD4+CD25+ nTreg cells was enhanced approximately twofold after stimulation with soluble anti-human CD3 (P < 0.005). Interestingly, the addition of LTA further enhanced the overall proliferation of CD4+CD25+ nTreg cells (P < 0.005). In contrast, a decrease (rather than an enhancement) in the absolute number of proliferating nTreg cells was observed when CpG2007 was used. 
Figure 3.
 
In vitro proliferation of CD4+CD25+ Treg cells purified from rabbit conjunctiva. CFSE-labeled CD4+CD25+ Treg cells were stimulated with 1 μg/mL soluble α-CD3 (A) or α-CD3 + APCs (B) for 6 days at 37°C in a 5% CO2 incubator, in the presence and absence of TLR ligand 2 (LTA) and TLR ligand 9 (CpG2007) at a concentration of 10 μg/mL and 1 μg/mL, respectively. Cells were harvested and stained with anti-rabbit CD4-PE or anti-rabbit CD25-PE and analyzed by flow cytometry. Values in each bar indicate the average ± SD of two wells. Each well has 5 × 104 CFSE-labeled CD4+CD25+ Treg cells and with and without 1 × 105 APCs (mitomycin C treated), as designated in the figure.
Figure 3.
 
In vitro proliferation of CD4+CD25+ Treg cells purified from rabbit conjunctiva. CFSE-labeled CD4+CD25+ Treg cells were stimulated with 1 μg/mL soluble α-CD3 (A) or α-CD3 + APCs (B) for 6 days at 37°C in a 5% CO2 incubator, in the presence and absence of TLR ligand 2 (LTA) and TLR ligand 9 (CpG2007) at a concentration of 10 μg/mL and 1 μg/mL, respectively. Cells were harvested and stained with anti-rabbit CD4-PE or anti-rabbit CD25-PE and analyzed by flow cytometry. Values in each bar indicate the average ± SD of two wells. Each well has 5 × 104 CFSE-labeled CD4+CD25+ Treg cells and with and without 1 × 105 APCs (mitomycin C treated), as designated in the figure.
Next we assessed whether the addition of autologous APCs would affect the proliferation of anti-CD3 stimulated nTreg cells. Purified CD4+CD25+ nTreg cells were stimulated in the presence of both soluble anti-CD3 and autologous APCs, in the presence of soluble anti-CD3 alone, or in the presence of media alone (control). As expected, the proliferation of nTreg was enhanced twofold to threefold when incubated with both anti-CD3 and autologous APCs compared with anti-CD3 without autologous APCs (Fig. 3B vs. Fig. 3A). The addition of LTA to these APC/CD4+CD25+ cocultures, along with anti-human CD3, further enhanced the proliferation by a factor of 10-fold compared with media alone (without anti-human CD3 or APCs). However, the addition of CpG2007, significantly decreased the overall proliferation of CD4+CD25+ nTreg cells, even in the presence of APCs (P < 0.005), Together these results demonstrate that TLR2 and TLR9 expressed by conjunctiva residual nTreg are functional. Although TLR2 ligand promoted nTreg proliferation, TLR9 ligand inhibited nTreg proliferation. 
TLR2 Ligand LTA Induces IFN-γ and IL-10 mRNA Expression in Conjunctiva-Resident CD4+CD25+ nTreg Cells
To determine whether conjunctiva-resident nTreg cells in “anergic” and “stimulated” states are able to produce IFN-γ, IL-10, and TGF-β, we checked their expression profiles by real-time PCR. Unfortunately, there are no kits available to detect rabbit cytokines and growth factor by ELISA. Conjunctiva-purified nTreg cells were pretreated with LTA for 6 hours, followed by incubation with anti-CD3 and IL-2 for another 6 hours at 37°C. Cells were harvested, and mRNA expression of IFN-γ, IL-10, and TGF-β were measured by real-time PCR. Figure 4 shows that LTA-pretreated and anti-CD3/IL-2–stimulated nTreg cells expressed 2.5-fold more IFN-γ (Fig. 4A) and IL-10 (Fig. 4B) mRNA than untreated nTreg cell controls (i.e., α CD3+ IL-2 without LTA; P < 0.05). However, LTA pretreatment did not affect TGF-β (Fig. 4C) expression because its mRNA level remained unchanged compared with untreated nTreg cell controls (i.e., α CD3+ IL-2 without LTA; P > 0.05). Treatment of nTreg cells with LTA alone, without CD3+ IL-2, was unable to induce significant levels of IFN-γ, IL-10, or TGF-β mRNAs compared with untreated nTreg cells (medium). These results suggest that LTA-TLR2 interaction might transform nTreg cells from an anergic state to an active state. Unlike LTA, CpG2007 treatment has no effect of on cytokine production by nTreg cells (Figs. 4D–F). 
Figure 4.
 
Quantification of INF-γ (A, D), IL-10 (B, E), and TGF-β (C, F) expression in rabbit conjunctiva-purified CD4+CD25+ cells using real-time PCR. One million nTreg cells were plated in a six-well tissue culture plate and were pretreated with LTA (10 μg/mL; AC) or CpG (1 μg/mL; DF) for 12 hours, followed by stimulation for another 12 hours in the presence of anti-CD3 (1 μg/mL) and IL-2 (20 U/mL) at 37°C. RNA was isolated from harvested cells and was quantified. An equal amount of RNA was reversed transcribed. cDNA was amplified using real time PCR and specific primers for rabbit INF-γ, IL-10, and TGF-β. GAPDH was used as a housekeeping gene. RNA quantification was calculated using the comparative Ct method, also known as the 2-ΔΔCt method, where ΔΔCt = ΔCt sample − ΔCt reference (none). Here, ΔCT sample is the Ct value for any sample normalized to the endogenous housekeeping gene, and ΔCt reference (none) is the Ct value for the calibrator also normalized to the endogenous housekeeping gene.
Figure 4.
 
Quantification of INF-γ (A, D), IL-10 (B, E), and TGF-β (C, F) expression in rabbit conjunctiva-purified CD4+CD25+ cells using real-time PCR. One million nTreg cells were plated in a six-well tissue culture plate and were pretreated with LTA (10 μg/mL; AC) or CpG (1 μg/mL; DF) for 12 hours, followed by stimulation for another 12 hours in the presence of anti-CD3 (1 μg/mL) and IL-2 (20 U/mL) at 37°C. RNA was isolated from harvested cells and was quantified. An equal amount of RNA was reversed transcribed. cDNA was amplified using real time PCR and specific primers for rabbit INF-γ, IL-10, and TGF-β. GAPDH was used as a housekeeping gene. RNA quantification was calculated using the comparative Ct method, also known as the 2-ΔΔCt method, where ΔΔCt = ΔCt sample − ΔCt reference (none). Here, ΔCT sample is the Ct value for any sample normalized to the endogenous housekeeping gene, and ΔCt reference (none) is the Ct value for the calibrator also normalized to the endogenous housekeeping gene.
Treatment with the TLR2 Ligand LTA Significantly Reverses the Immunosuppressive Function of CD4+CD25+ nTreg Cells
We next assessed the effect of TLR2 and TLR9 ligands on the immunosuppressive function of conjunctiva-resident nTreg cells. In a conventional in vitro suppression assay, purified CD4+CD25 Teff cells were first labeled with CFSE and then incubated with purified nonlabeled CD4+CD25+ nTreg cells in the presence or absence of either TLR2 (LTA) or TLR9 (CpG2007) ligands. As shown in Figure 5A, the addition of CD4+CD25+ nTreg cells at a ratio of 1:2 to CD4+CD25 Teff cells resulted in a significant suppression of CD4+CD25 Teff cell proliferation (P < 0.005). The results presented in Figure 5A were also expressed as the percentage of proliferation in Figure 5B. This CFSE-labeled suppression assay result confirmed our previous observation 1 that conjunctiva-resident nTreg cells can suppress the proliferation of CD4+CD25 Teff cells. Here we observed approximately 92% suppression of the proliferation of CD4+CD25 Teff cells in the presence of nTreg cells. Interestingly, when LTA was added in the Teff and nTreg coculture cells, the suppressive effect of nTreg cells was reversed, and the CD4+CD25 Teff cells regained their proliferation efficiency by an average of 30%. However, under identical conditions, the addition of CpG2007 to the Teff and nTreg coculture cells reversed the suppression effect by only 10%. This result suggests that TLR2 ligand has the potential to reverse the suppressive property of CD4+CD25+ nTreg cells. Under these experimental conditions, LTA was more efficient than CpG2007 in reversing the suppressive function of CD4+CD25+ nTreg cells. 
Figure 5.
 
In vitro suppression of CD+CD25 effector cells by CD4+CD25+ nTreg. Conjunctiva-purified CD+CD25 effector cells (5 × 104 cells) were labeled with CFSE and stimulated with soluble α human CD3 (1 μg/mL) and mitomycin C (50 μg/mL)–treated autologous APCs (1 × 105 cells) for 5 days at 37°C. For the suppression assay, CFSE-labeled CD4+CD25 effector cells (5 × 104 cells) were mixed with nonlabeled CD4+CD25+ cells (10 × 104 cells) in culture medium containing soluble α human CD3 (1 μg/mL) and autologous APCs (1 × 105 cells) and were incubated in the presence and absence of LTA (10 μg/mL) and CpG2007 (1 μg/mL) for 5 days at 37°C. Cells were harvested and stained with α rabbit CD4-PE and analyzed by flow cytometry. (A, dotted line) Absolute number of CD4+CD25 effector cells in the absence of anti-human CD3 and autologous APCs. (B) Percentage of Teff cells calculated from (A). Values in each bar indicate the average ± SD of two wells. (C) Histogram that represents nonstimulated CFSE-labeled CD4+CD25 Teff cells (dashed lines) overlaid with histograms (bold lines) that represent CFSE-labeled CD4+CD25 Teff cells stimulated with (a) an anti-CD3+ mAb alone or with (b) an anti-CD3+ mAb in the presence of untreated CD4+CD25+ Treg cells, (c) an anti-CD3+ mAb in the presence of LTA-treated CD4+CD25+ Treg cells, or (d) an anti-CD3+ mAb in the presence of CpG-treated CD4+CD25+ Treg cells.
Figure 5.
 
In vitro suppression of CD+CD25 effector cells by CD4+CD25+ nTreg. Conjunctiva-purified CD+CD25 effector cells (5 × 104 cells) were labeled with CFSE and stimulated with soluble α human CD3 (1 μg/mL) and mitomycin C (50 μg/mL)–treated autologous APCs (1 × 105 cells) for 5 days at 37°C. For the suppression assay, CFSE-labeled CD4+CD25 effector cells (5 × 104 cells) were mixed with nonlabeled CD4+CD25+ cells (10 × 104 cells) in culture medium containing soluble α human CD3 (1 μg/mL) and autologous APCs (1 × 105 cells) and were incubated in the presence and absence of LTA (10 μg/mL) and CpG2007 (1 μg/mL) for 5 days at 37°C. Cells were harvested and stained with α rabbit CD4-PE and analyzed by flow cytometry. (A, dotted line) Absolute number of CD4+CD25 effector cells in the absence of anti-human CD3 and autologous APCs. (B) Percentage of Teff cells calculated from (A). Values in each bar indicate the average ± SD of two wells. (C) Histogram that represents nonstimulated CFSE-labeled CD4+CD25 Teff cells (dashed lines) overlaid with histograms (bold lines) that represent CFSE-labeled CD4+CD25 Teff cells stimulated with (a) an anti-CD3+ mAb alone or with (b) an anti-CD3+ mAb in the presence of untreated CD4+CD25+ Treg cells, (c) an anti-CD3+ mAb in the presence of LTA-treated CD4+CD25+ Treg cells, or (d) an anti-CD3+ mAb in the presence of CpG-treated CD4+CD25+ Treg cells.
TLR2 Ligand LTA Enhances the Immunogenicity of HSV-1 gD–Derived Peptide Antigen Delivered Topically to the Conjunctiva
Next we compared the adjuvant effect of TLR2 and TLR9 ligands on topical ocular mucosal immunogenicity of the HSV-1 gD peptide epitope. Rabbits were immunized by topical application of a mixture of four immunodominant CD4+ T cell peptide epitopes (gD144–179, gD287–317, gD49–82, gD332–358) selected from HSV gD, 36,37 along with either the TLR2 ligand LTA or the TLR9 ligand CpG2007 as immunoadjuvants. The control group received saline alone, LTA alone, or CpG2007 alone (Mock). After the third immunization, conjunctivas were harvested from each immunized and mock-immunized rabbit group. Single cell suspension was labeled with CFSE, and the proliferation of CD4+CD25 Teff cells was monitored following stimulation with the individual HSV gD peptide (i.e., gD144–179, gD287–317, gD49–82, or gD332–358). The cells were analyzed by flow cytometry on gated CD4+CD25 Teff cells. Figure 6 shows a comparative analysis of the proliferation of CD4+ T cells obtained from gD peptides + LTA versus gD peptides + CpG2007-immunized rabbits. Significant CD4+CD25 Teff cell proliferation was obtained against gD287–317- and gD49–82-immunodominant peptide epitopes when LTA was used as an immunoadjuvant compared with when CpG2007 was used as an immunoadjuvant (P < 0.005). However, no significant difference in T cell proliferative responses against gD144–179 and gD49–82 peptides were detected in the CpG2007 and LTA groups. 
Figure 6.
 
Six rabbits (two per group) were immunized ocularly three times at an interval of 14 days with a mixture of four HSV-gD peptide (gD144–179, gD287–317, gD49–82, and gD332–358) mixed with either CpG2007 (group 1) or LTA (group 2) as a topical ocular mucosal immunoadjuvants. The control group received saline alone, LTA alone, or CpG2007 alone (mock, group 3). Ten days after the third immunization, rabbits were euthanatized, upper and lower conjunctivas were harvested from each rabbit, and lymphocytes were isolated. Lymphocytes were labeled with CFSE and stimulated with HSV-gD peptide (gD144–179, gD287–317, gD49–82, or gD332–358)–pulsed autologous APCs for 5 days at 37°C in a CO2 incubator. Cells were harvested and stained with anti-rabbit CD4-PE, and their proliferation was analyzed by flow cytometry. (A) Absolute number of CFSE-labeled proliferating CD4+ cells under the marked conditions. (B) Dot plot representation with the percentage of CFSE-labeled proliferating CD4+ cells.
Figure 6.
 
Six rabbits (two per group) were immunized ocularly three times at an interval of 14 days with a mixture of four HSV-gD peptide (gD144–179, gD287–317, gD49–82, and gD332–358) mixed with either CpG2007 (group 1) or LTA (group 2) as a topical ocular mucosal immunoadjuvants. The control group received saline alone, LTA alone, or CpG2007 alone (mock, group 3). Ten days after the third immunization, rabbits were euthanatized, upper and lower conjunctivas were harvested from each rabbit, and lymphocytes were isolated. Lymphocytes were labeled with CFSE and stimulated with HSV-gD peptide (gD144–179, gD287–317, gD49–82, or gD332–358)–pulsed autologous APCs for 5 days at 37°C in a CO2 incubator. Cells were harvested and stained with anti-rabbit CD4-PE, and their proliferation was analyzed by flow cytometry. (A) Absolute number of CFSE-labeled proliferating CD4+ cells under the marked conditions. (B) Dot plot representation with the percentage of CFSE-labeled proliferating CD4+ cells.
TLR2 Ligand Pam3CSK4, but Not LTA, Affects Foxp3 Expression in Conjunctiva-Resident CD4+CD25+ nTreg Cells
We investigated whether treatment with TLR2 ligands (LTA and Pam3CSK4) or TLR9 ligand (CpG) would affect Foxp3 expression in rabbit conjunctiva CD4+CD25+ Treg cells. A slight increase in Foxp3 expression was detected, by both flow cytometry (Figs. 7A, 7B) and Western blot analysis (Fig. 7C), after CpG treatment. However, no significant changes in the Foxp3 level were observed after LTA treatment. Interestingly, treatment of rabbit conjunctiva CD4+CD25+ Treg cells with a different TLR2 ligand, Pam3CSK4, induced a significant decrease in the level of Foxp3 (P < 0.005). 
Figure 7.
 
Foxp3 expression on rabbit conjunctival Treg by TLR2 and TLR9 ligands. Rabbit conjunctiva–purified CD4+CD25+ Treg cells (0.5 × 106 cells) were either left untreated or stimulated overnight with anti-human CD3 (1 μg/mL) ± treatment with LTA (10 μg/mL), CpG (1 μg/mL), or Pam3CSK4 (50 ng/mL). TLR ligand treatments and anti-CD3 stimulations were performed together in a 90-well, flat-bottomed tissue culture plate at 37°C/5% CO2 incubator for overnight. (A, B), Cells were harvested and surface stained with anti-rabbit CD4-FITC (10 μL/assay) followed by intracellular staining with anti-human Foxp3-PE (20 μL/assay) and were analyzed by FACS. (C) Cells from each well (identical to those described) were lysed with 100-μL lysis buffer (RIPA with PI cocktail). Equal volumes (20 μL) of denatured cell lysates containing ∼1 × 105 cells were loaded in each lane. Details of the Western blot procedure have been previously described. 1 (A, dot plot) MFI of CD4+/Foxp3-positive cells for rabbit conjunctiva-purified Treg cells (top) and human PBMC-purified Treg cells (bottom). Human PBMC-purified Treg cells were used as a positive control in this assay. (B, overlapped histograms) Expression of Foxp3 in Treg by FACS. (C) Foxp3 protein in rabbit conjunctiva-purified CD4+CD25+ Treg cells detected by Western blot analysis.
Figure 7.
 
Foxp3 expression on rabbit conjunctival Treg by TLR2 and TLR9 ligands. Rabbit conjunctiva–purified CD4+CD25+ Treg cells (0.5 × 106 cells) were either left untreated or stimulated overnight with anti-human CD3 (1 μg/mL) ± treatment with LTA (10 μg/mL), CpG (1 μg/mL), or Pam3CSK4 (50 ng/mL). TLR ligand treatments and anti-CD3 stimulations were performed together in a 90-well, flat-bottomed tissue culture plate at 37°C/5% CO2 incubator for overnight. (A, B), Cells were harvested and surface stained with anti-rabbit CD4-FITC (10 μL/assay) followed by intracellular staining with anti-human Foxp3-PE (20 μL/assay) and were analyzed by FACS. (C) Cells from each well (identical to those described) were lysed with 100-μL lysis buffer (RIPA with PI cocktail). Equal volumes (20 μL) of denatured cell lysates containing ∼1 × 105 cells were loaded in each lane. Details of the Western blot procedure have been previously described. 1 (A, dot plot) MFI of CD4+/Foxp3-positive cells for rabbit conjunctiva-purified Treg cells (top) and human PBMC-purified Treg cells (bottom). Human PBMC-purified Treg cells were used as a positive control in this assay. (B, overlapped histograms) Expression of Foxp3 in Treg by FACS. (C) Foxp3 protein in rabbit conjunctiva-purified CD4+CD25+ Treg cells detected by Western blot analysis.
Discussion
It was initially thought that TLRs are primarily expressed by APCs, such as macrophages and dendritic cells, and that interactions between microbial ligands and TLRs expressed by APCs would indirectly result in activation of effector T cells. However, it has now become evident that TLRs are also expressed by various T cell subsets, such as conventional αβ Teff cells, Treg cells, and γδ T cells and by natural killer T cells. 38 Importantly, it appears that at least in some of these T cell subsets, TLRs are functionally active because stimulation of these cells with TLR agonists in the absence of APCs results in the exertion of effector or regulatory functions of T cells. In the present study, we provided evidence for the dominating expression of TLR2 and TLR9, over other TLRs, by conjunctiva-resident nTreg cells and demonstrated their functional relevance with respect to ocular mucosal T cell immunity. To our knowledge, this is the first report describing the phenotypic expression and functional property of TLRs by conjunctiva-resident T cells. 
Most data on mucosal Treg cells originated from mouse models (see Ref. 12 for review). Because of the obvious ethical and practical considerations in studying human ocular mucosal Treg cells, a critical question has been which species would be the most appropriate animal model to investigate conjunctiva Treg cells. 39,40 Instead of mice we elected rabbit as the working animal model, bearing in mind that numerous similarities exist between rabbit and human ocular mucosal tissues 40 42 ; several T cell–mediated ocular surface diseases, including herpetic conjunctivitis and recurrent corneal herpetic stromal keratitis (HSK), have been reported to be similar in rabbits and humans, making it the most relevant animal model for exploring these human eye disorders 43 45 ; compared with mice, rabbit conjunctiva-associated lymphoid tissue (CALT) more closely resembles human CALT 46 48 ; microanatomy and immunohistologic studies indicate that rabbit conjunctival mucosa is comparable to that of humans and has a typical follicular ultrastructure with an abundance of conjunctival lymphoid follicles, whereas no lymphoid tissue was identified in mice and rats 48 52 ; from a practical standpoint, unlike mice, rabbits have a relatively large conjunctival surface, offering abundant MALT for in vitro studies; finally, the recent availability of many monoclonal and polyclonal antibodies specific to rabbit immune cell CD markers, cytokines, and growth factors provides useful immunologic tools for an unprecedented phenotypic and functional analysis of rabbit T cell repertoire and function. 
Rabbit conjunctiva-purified CD4+CD25+ Treg cells display an increased level of TLR expression, especially TLR2 compared with autologous CD4+CD25 Teff cells, suggesting that the expansion and suppression function of Treg cells may be closely regulated by TLR2 ligands. In general, Treg cells express higher levels of TLR2, TLR4, TLR5, TLR7/8, and TLR10 than conventional CD4+CD25 Teff cells. 28 31,34,38,53,54 TLR10 is constitutively expressed at both mRNA and protein levels in Treg cells but not in Teff cells. 55 On the other hand, TLR3 is not detectable in Treg cells, whereas it is expressed by Teff cells. 38,56 Several recent reports indicate direct sensing of “danger” signals by T cells (instead of APCs). This direct recognition of TLR ligands may play a role in T cell homeostasis and effector function. In particular, a crucial role of TLR2 activation in the functional properties of Treg cells has been demonstrated in a series of reports showing that TLR2 ligand temporarily abrogates the immunosuppressive effects of murine Treg cells and reduces the level of Foxp3 expression in Treg cells and activates the expansion of dysfunctional Treg cells. 30,34,38 Here we found that engagement of TLR2 on rabbit conjunctival Treg cells with its specific ligand, LTA, induced their proliferation (Fig. 3) and reversed their immunosuppressive function (Fig. 5). Questions might arise whether this downregulation of immunosuppression effect is due to an engagement of LTA on Treg cells or a direct effect on Teff. Although we cannot rule out the latter possibility, the probability is low because autologous rabbit Teff cells express fivefold to sixfold less TLR2 than Treg cells (Fig. 1D). Furthermore, in a separate experiment, we have shown that LTA does not affect the proliferation of CD4+CD25 Teff cells in the presence of soluble anti-CD3 and autologous APCs (data not shown). In contrast to TLR-2, the expression level of TLR9 on both Treg and Teff cells is more or less similar (Figs. 1C, 1D). In the Treg cell proliferation assay, when TLR9 is engaged with the specific ligand CpG, we observed a significant inhibition of Treg cell proliferation (Fig. 3). However, in the suppression assay (Fig. 5), CpG reversed the immunosuppressive capability of Treg cells, but at lesser extent than LTA. Whether this reduced effect of CpG in the suppression assay was caused by a simultaneous engagement of TLR9 on both Treg or Teff cells remains to be determined. Nevertheless, in a separate experiment, we have shown that CpG did not affect the proliferation of CD4+CD25 Teff cells in the presence of soluble anti-CD3 and autologous APCs, thus excluding a direct effect on Teff cells (data not shown). Our results are consistent with recent finding by Chiffoleau et al. 57 showing that triggering TLR9 on human Treg cells inhibits their immunosuppressive function but does not induce their proliferation or the downregulation of their Foxp3 expression. In that study the loss of suppression was in part due to the effect of TLR9 ligand on Teff cells, but it is not clear whether TLR9 ligand directly influences Treg cells. 
In this study we found that conjunctiva-resident CD4+ CD25+ nTreg cells express relatively high levels of TLR2 (MFI ∼260), a moderate level of TLR9 (MFI ∼115), and very low levels of TLR3, TLR4, and TLR8 (MFI <50). In parallel, we checked the expression profile of the same set of TLRs in the copurified fraction of CD4+CD25 Teff cells and found extremely low levels (MFI <50) for TLR3, TLR4, and TLR8 and a relatively higher level of TLR9 (MFI ∼88) expression. This indicated that, with the exception of TLR9, the other TLRs expression on Teff cells was statistically insignificant in the ocular mucosal conjunctiva. To determine whether the expression level of TLRs on conjunctiva-resident nTreg cells was comparable with other immune cells known for high TLR expression, we evaluated the TLR expression profile in autologous APCs. Our data show that conjunctiva-resident CD11b+ APCs follow similar expression patterns for TLR2 (MFI ∼260) and low levels for TLR3, TLR4, and TLR8. However, the TLR9 level was extremely high and was expressed four to five times more (MFI ≥500) than the expression of TLR9 (MFI ≥100) on nTreg cells. It is interesting that though a differential level of TLR9 expression was observed in two different immune cells, such as CD11b+ APCs and nTreg cells of same conjunctival origin, the TLR2 expression level remained similar in both types of cells. This suggests that TLR2 is a more conserved type of toll receptor within the immune cells present in the mucosal conjunctiva tissue. However, in humans and mice, TLR4, TLR5, and TLR8, but not TLR2, are preferentially expressed on CD4+CD25+ Treg cells. 28 31,34,38,53,54 This highlights a striking phenotypic difference between mucosa and non-mucosa nTreg cells. A similar observation of TLR2 and TLR9 expression in ocular epithelial cells was reported by Chang et al. 58 To our knowledge, there are thus far two reports of TLR2 expression on CD4+CD25+ Treg cells in humans and mice. 30,33 Although TLR expression patterns and magnitudes varied within the tissue and species, 59,60 in rabbit, the selective high expression of TLR2 on conjunctiva-resident nTreg cells is certainly a unique situation. 
It has been shown that TLR ligands influence the immunosuppressive function of nTreg cells either through a direct interaction with TLRs specifically expressed on Treg cells 31 or an indirect mechanism mediated by dendritic cells. 61 In this study, TLR2 ligand LTA induced in vitro the proliferation of conjunctiva-resident Treg cells in the presence of APCs and partially inhibited the immunosuppressive function of nTreg cells. LTA alone failed to induce proliferation of Treg cells. Interestingly, under identical experimental conditions, despite the moderate level of TLR9 expression on conjunctiva-resident Treg cells, we found a strong inhibitory effect on Treg cell proliferation and immunosuppressive function in vitro in the presence of the TLR9 ligand CpG2007. Accordingly, our in vivo data reflected that reducing Treg cell proliferation by CpG2007 increased the proliferation of rabbit ocular CD4+ Teff cells specific to HSV-1 gD peptides. In contrast to CpG2007, treatment of rabbit eyes by the TLR2 ligand (LTA) increases (i) the proliferation of Treg cells, (ii) the mRNA level of IL-10 and IFN-γ and (iii) partially abrogates Treg cell immunosuppressive function. This suggests that although LTA increases the pool of the Treg population, those newly proliferated Treg cells are functionally different from the original ones. These LTA-proliferated Treg cells not only lost their suppressive role by 30% (Fig 5B), they also expressing higher levels of IL-10 and IFN-γ mRNA, which is not conventional for nTreg cells. A similar effect was reported by Sutmuller et al. 30,33 The TLR2 ligand Pam3Cys-SK4 forced Treg cells into the proliferative pathway, which was paralleled by a transient loss of suppressive activity, and they regained their suppression once cells were back in the resting phase. 30,33 One possible explanation for the loss of suppression by LTA would be the higher production of IFN-γ by nTreg cells when stimulated in the presence of LTA/anti-CD3/IL-2 (Fig. 4A). We have also detected higher expression of TGF-β in nTreg cultures stimulated with LTA/anti CD3/IL-2 (Fig. 4C), thus confirming previous reports. 62 However, controversial data about the role of TGF-β in nTreg cells have also been reported. 63  
The most prominent characteristics of nTreg cells are their nonresponsiveness toward TCR stimulation in the presence of anti-CD3. However, our in vitro data (Fig. 3A) showed that the proliferation of nTreg cells was slightly increased by anti-CD3 irrespective of the presence of either LTA or CpG or the addition of APCs in the culture. This suggested that nTreg cells might not be equally anergic in all tissues and species. HSV infections occur at the ocular mucosal tissues and lead to self-limiting primary disease. 64 Foreign HSV antigens promote immune responses when coadministered with TLR ligands, which have been exploited as immune adjuvants promoting protective T cells and antibody responses. 65,66 Here we compared the potential efficacy of LTA and CpG as candidate immune adjuvants in the rabbit eye model and showed that although both CpG2007 and LTA are capable of inducing local CD4+ Teff cell responses, these responses were better in the presence of LTA. Collectively, these results strongly suggest that TLR2 signals contribute to the activation and expansion of nTreg cells (Fig. 3) and interfere with their suppressive activity (Fig. 5). Consequently, this led to an increase the magnitude of HSV-gD-peptide–specific Teff cell responses in vivo (Fig. 6). As suggested by Conroy et al. 67 the nTreg cell function is more like a “double-edged sword” that can lead to an outcome that is context dependent. Under these circumstances, greater understanding of the modulation and regulation of Treg cells in the rabbit ocular immune system is critical to understand their complete biological role. 
It has been shown that though TLR2, TLR8, or TLR9 ligation abrogates or reverses the immunosuppressive function of CD4+CD25+ Treg cells, TLR2, TLR4, or TLR5 ligation enhances the suppressive capacity of CD4+CD25+ Treg cells. 68 Leu et al. 33 recently showed that in the presence of a TLR2 agonist, such as the synthetic bacterial lipoprotein Pam3Cys-SK4, there was marked expansion of CD4+CD25+ Treg cells, but their immunosuppressive function was temporarily abrogated. This study suggests that the downregulation of Foxp3 could be a putative mechanism for the abrogation of Treg cell–mediated suppression. 33,69 Treatment of CD4+CD25+ Treg cells with heat shock protein 60 (the in vivo ligand of TLR2) suppressed target Teff cells both by cell-cell contact mechanisms and by the secretion of cytokines such as TGF-β and IL-10. 70 TLR2 activation on Treg cells using the synthetic ligand PAM3Cys, in combination with IL-2 and TCR triggering, can induce Treg proliferation and results in temporary loss of suppression. On removal of the TLR2 ligand, the Treg cells regained their immunosuppressive function. 27,30,71,72 This discrepancy could possibly be explained by the nature of the TLR ligands used (PAM3Cys is an exogenous TLR1/2 ligand and hsp60 is an endogenous TLR2 ligand), the concentrations of the ligands used, or differences in the ways endogenous and exogenous ligands interact with TLR2. TLR8 also directly reverses the immunosuppressive function of CD4+CD25+ Treg cells. 29 TLR8 is strongly and preferentially expressed on human Treg cells compared with human Teff cells. 29 Triggering TLR8 on Treg cells resulted in the specific abrogation of suppression without affecting Treg cell proliferation, though no effects on human Teff cells were observed. 29 Applying siRNA technology to knockdown TLR8 on Treg cells completely blocked the effect, demonstrating a crucial role for TLR8. 29  
Several studies, using mouse- or human-derived CD4+ CD25+ Treg, pointed to an interplay between TLRs and Foxp3. 73 In humans, the engagement of TLR5 by its ligand flagellin leads to the upregulation of Foxp3 expression in Treg and consequently enhances their suppressive activity. 28,74,75 In contrast, TLR9 engagement by CpG did not affect Foxp3 expression in Treg although it abrogated their suppressive function. 32,57,76 Although the effect of most TLR engagement on Foxp3 expression and Treg-suppressive function appears to be settled for many TLRs, conflicting results have been reported in mice on the effect of TLR2 engagement on Foxp3 expression on Treg. Some studies showed that Foxp3 expression was downregulated after TLR2 ligation by the synthetic bacterial triacylated lipopeptide Pam3CSK4. 30,33 Chen et al. 77 recently challenged this report by showing that treatment of Treg with Pam3CSK4 neither affected the level of Foxp3 expression on Treg cells nor abolished its suppressive function. This discrepancy could be explained by the use of different TLR2 ligands in each study that might lead to the use of different heterodimers, the possibility that there are species-specific differences in the control of Treg cell activity by same TLR2 ligands, and the fact that differences might be linked to the origin of Treg in each study (i.e., peripheral vs. mucosal-derived Treg). In contrast to the murine system, TLR2 ligand–mediated abrogation of human Treg function was not associated with a downregulation of Foxp3. 73 Nevertheless, our data (Fig. 7) showed some increase in Foxp3 expression when TLR9 was engaged with CpG. However, no significant changes in Foxp3 expression were observed when TLR2 was engaged with LTA. Interestingly, the engagement of TLR2 with a different ligand Pam3CSK4 significantly decreased the Foxp3 expression on rabbit Treg. 
In humans, the LTA ligand engages the TLR2/TLR6 combination, whereas Pam3Cys engages TLR2/TLR1. 78 In mice, the Pam3CSK4 ligand engages either the TLR2/TLR1 combination or TLR2 independently of TLR1. Furthermore, Hajjar et al. 79 reported on the recognition of triacylated lipopeptides in a TLR2/TLR1-dependent fashion in human cells. In the presence of the Pam3CSK4 ligand, a stable heterodimer is formed between TLR2 and TLR1. 80 Therefore, though both LTA and Pam3CSK4 are ligands for TLR2, the former engages TLR2/TLR6 and the latter engages TLR2/TLR1 combinations. Nevertheless, recent data by Carpenter and colleagues 81 recently showed that engagement of either TLR2/TLR1 or TLR2/TLR6 combinations activate the same NF-κB downstream signal pathway. To our knowledge, the present study is first to report the expression and functional engagement of any TLR on rabbit Treg cells. We demonstrated that engagement of TLR-2 on rabbit Treg cells with LTA and Pam3CSK4 differentially affects the expression of Foxp3 (Fig. 7), suggesting that, as with human Treg cells, these ligands might interact with either TLR2/TLR1 or TLR2/TLR6 combination. Given that most of the TLR data come from mice and humans, there is a possibility that LTA and Pam3CSK4 ligands might have different effects on rabbit TLR2/TLR-1 and TLR2/TLR-6 combinations and downstream intracellular pathways. This will be the subject of future reports. 
In conclusion, the results reported here suggest a link between the TLR2 and its exogenous ligand LTA and how nTreg cells in ocular mucosal tissue regulate cellular immunity in response to viral antigen. These findings might be useful in clinical settings to enhance the efficacy of immunotherapy directed toward infectious diseases. CD4+ T cell help is required for the generation of primary CTL responses and the promotion of protective CD8+ memory T cell development. Recent studies 82,83 demonstrated a previously unappreciated role of CD4 help in mobilizing effector CD8+ CTL to the peripheral sites of infection. Our results reveal a previously unappreciated role of CD4+CD25+ nTreg cells in interfering with CD4+CD25 Teff cells at the ocular mucosal sites of herpes infection, where they might help to eliminate herpes-infected cells. Ongoing and future research will further delineate the mechanisms involved in interactions between these two subsets of T cells and innate receptors and will pave the way for developing better prophylactic or therapeutic strategies for control of infectious, allergic, and autoimmune ocular diseases. 
Footnotes
 Supported by Public Health Service Grants EY014900 and EY019896 (LBM), the Discovery Eye Foundation, and a Research to Prevent Blindness (RPB) Challenge Grant. LBM is an RPB Special Award Investigator.
Footnotes
 Disclosure: G. Dasgupta, None; A.A. Chentoufi, None; S. You, None; P. Falatoonzadeh, None; L.A.A. Urbano, None; A. Akhtarmalik, None; K. Nguyen, None; L. Ablabutyan, None; A.B. Nesburn, None; L. BenMohamed, None
References
Nesburn AB Bettahi I Dasgupta G . Functional Foxp3+ CD4+ CD25(Bright+) “natural” regulatory T cells are abundant in rabbit conjunctiva and suppress virus-specific CD4+ and CD8+ effector T cells during ocular herpes infection. J Virol. 2007;81:7647–7661. [CrossRef] [PubMed]
Belkaid Y . Regulatory T cells and infection: a dangerous necessity. Nat Rev Immunol. 2007;7:875–888. [CrossRef] [PubMed]
Taylor PA Noelle RJ Blazar BR . CD4(+)CD25(+) immune regulatory cells are required for induction of tolerance to alloantigen via costimulatory blockade. J Exp Med. 2001;193:1311–1318. [CrossRef] [PubMed]
Lewkowich IP Herman NS Schleifer KW . CD4+CD25+ T cells protect against experimentally induced asthma and alter pulmonary dendritic cell phenotype and function. J Exp Med. 2005;202:1549–1561. [CrossRef] [PubMed]
Iwashiro M Messer RJ Peterson KE Stromnes IM Sugie T Hasenkrug KJ . Immunosuppression by CD4+ regulatory T cells induced by chronic retroviral infection. Proc Natl Acad Sci U S A. 2001;98:9226–9230. [CrossRef] [PubMed]
Belkaid Y . Role of Foxp3-positive regulatory T cells during infection. Eur J Immunol. 2008;38:918–921. [CrossRef] [PubMed]
Belkaid Y Oldenhove G . Tuning microenvironments: induction of regulatory T cells by dendritic cells. Immunity. 2008;29:362–371. [CrossRef] [PubMed]
Dejaco C Duftner C Grubeck-Loebenstein B Schirmer M . Imbalance of regulatory T cells in human autoimmune diseases. Immunology. 2006;117:289–300. [CrossRef] [PubMed]
Randolph DA Fathman CG . CD4+CD25+ regulatory T cells and their therapeutic potential. Annu Rev Med. 2006;57:381–402. [CrossRef] [PubMed]
Shimizu J Yamazaki S Takahashi T Ishida Y Sakaguchi S . Stimulation of CD25(+)CD4(+) regulatory T cells through GITR breaks immunological self-tolerance. Nat Immunol. 2002;3:135–142. [CrossRef] [PubMed]
Shevach EM McHugh RS Piccirillo CA Thornton AM . Control of T cell activation by CD4+ CD25+ suppressor T cells. Immunol Rev. 2001;182:58–67. [CrossRef] [PubMed]
Wohlfert E Belkaid Y . Plasticity of T reg at infected sites. Mucosal Immunol. 2010;3:213–215. [CrossRef] [PubMed]
Toka FN Suvas S Rouse BT . CD4+ CD25+ T cells regulate vaccine-generated primary and memory CD8+ T cell responses against herpes simplex virus type 1. J Virol. 2004;78:13082–13089. [CrossRef] [PubMed]
Wildin RS Ramsdell F Peake J . X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy. Nat Genet. 2001;27:18–20. [CrossRef] [PubMed]
Bennett CL Christie J Ramsdell F . The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat Genet. 2001;27:20–21. [CrossRef] [PubMed]
Brunkow ME Jeffery EW Hjerrild KA . Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat Genet. 2001;27:68–73. [CrossRef] [PubMed]
Redfern RL McDermott AM . Toll-like receptors in ocular surface disease. Exp Eye Res. 2010;90:679–687. [CrossRef] [PubMed]
Iwasaki A Medzhitov R . Regulation of adaptive immunity by the innate immune system. Science. 2010;327:291–295. [CrossRef] [PubMed]
Gelman AE Zhang J Choi Y Turka LA . Toll-like receptor ligands directly promote activated CD4+ T cell survival. J Immunol. 2004;172:6065–6073. [CrossRef] [PubMed]
Kumar A Singh CN Glybina IV Mahmoud TH Yu FS . Toll-like receptor 2 ligand-induced protection against bacterial endophthalmitis. J Infect Dis. 2010;201:255–263. [CrossRef] [PubMed]
Johnson AC Heinzel FP Diaconu E . Activation of toll-like receptor (TLR)2, TLR4, and TLR9 in the mammalian cornea induces MyD88-dependent corneal inflammation. Invest Ophthalmol Vis Sci. 2005;46:589–595. [CrossRef] [PubMed]
Tabeta K Georgel P Janssen E . Toll-like receptors 9 and 3 as essential components of innate immune defense against mouse cytomegalovirus infection. Proc Natl Acad Sci U S A. 2004;101:3516–3521. [CrossRef] [PubMed]
Ishii KJ Coban C Akira S . Manifold mechanisms of toll-like receptor-ligand recognition. J Clin Immunol. 2005;25:511–521. [CrossRef] [PubMed]
Johnson A Pearlman E . Toll-like receptors in the cornea. Ocul Surf. 2005;3:S187–S189. [PubMed]
Sun Y Hise AG Kalsow CM Pearlman E . Staphylococcus aureus-induced corneal inflammation is dependent on Toll-like receptor 2 and myeloid differentiation factor 88. Infect Immun. 2006;74:5325–5332. [CrossRef] [PubMed]
Aderem A Ulevitch RJ . Toll-like receptors in the induction of the innate immune response. Nature. 2000;406:782–787. [CrossRef] [PubMed]
Netea MG Sutmuller R Hermann C . Toll-like receptor 2 suppresses immunity against Candida albicans through induction of IL-10 and regulatory T cells. J Immunol. 2004;172:3712–3718. [CrossRef] [PubMed]
Crellin NK Garcia RV Hadisfar O Allan SE Steiner TS Levings MK . 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. 2005;175:8051–8059. [CrossRef] [PubMed]
Peng G Guo Z Kiniwa Y . Toll-like receptor 8-mediated reversal of CD4+ regulatory T cell function. Science. 2005;309:1380–1384. [CrossRef] [PubMed]
Sutmuller RP den Brok MH Kramer M . Toll-like receptor 2 controls expansion and function of regulatory T cells. J Clin Invest. 2006;116:485–494. [CrossRef] [PubMed]
Caramalho I Lopes-Carvalho T Ostler D Zelenay S Haury M Demengeot J . Regulatory T cells selectively express toll-like receptors and are activated by lipopolysaccharide. J Exp Med. 2003;197:403–411. [CrossRef] [PubMed]
Chiffoleau E Heslan JM Heslan M Louvet C Condamine T Cuturi MC . TLR9 ligand enhances proliferation of rat CD4+ T cell and modulates suppressive activity mediated by CD4+ CD25+ T cell. Int Immunol. 2007;19:193–201. [CrossRef] [PubMed]
Liu H Komai-Koma M Xu D Liew FY . Toll-like receptor 2 signaling modulates the functions of CD4+ CD25+ regulatory T cells. Proc Natl Acad Sci U S A. 2006;103:7048–7053. [CrossRef] [PubMed]
Sutmuller RP Morgan ME Netea MG Grauer O Adema GJ . Toll-like receptors on regulatory T cells: expanding immune regulation. Trends Immunol. 2006;27:387–393. [CrossRef] [PubMed]
Rozen S Skaletsky HJ . Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S Misener S eds. Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, NJ: 2000;365–386.Source code available at http://fokker.wi.mit.edu/primer3/ .
Chentoufi AA Binder NR Berka N . Asymptomatic human CD4+ cytotoxic T cell epitopes identified from herpes simplex virus glycoprotein B. J Virol. 2008;83:11792–11802. [CrossRef]
Chentoufi AA Zhang X Lamberth K . HLA-A*0201-restricted CD8+ cytotoxic T lymphocyte epitopes identified from herpes simplex virus glycoprotein D. J Immunol. 2008;180:426–437. [CrossRef] [PubMed]
Kulkarni R Behboudi S Sharif S . Insights into the role of Toll-like receptors in modulation of T cell responses. Cell Tissue Res. 2010;343:141–152. [CrossRef] [PubMed]
Gebert A Pabst R . M cells at locations outside the gut. Semin Immunol. 1999;11:165–170. [CrossRef] [PubMed]
Chodosh J Kennedy RC . The conjunctival lymphoid follicle in mucosal immunology. DNA Cell Biol. 2002;21:421–433. [CrossRef] [PubMed]
Romanowski EG Araullo-Cruz T Gordon YJ . Topical corticosteroids reverse the antiviral effect of topical cidofovir in the Ad5-inoculated New Zealand rabbit ocular model. Invest Ophthalmol Vis Sci. 1997;38:253–257. [PubMed]
Romanowski EG Gordon YJ Araullo-Cruz T Yates KA Kinchington PR . The antiviral resistance and replication of cidofovir-resistant adenovirus variants in the New Zealand White rabbit ocular model. Invest Ophthalmol Vis Sci. 2001;42:1812–1815. [PubMed]
Nesburn AB Dunkel ED Trousdale MD . Enhanced HSV recovery from neuronal tissues of latently infected rabbit. Proc Soc Exp Biol Med. 1980;163:398–401. [CrossRef] [PubMed]
Nesburn AB . Common viral eye diseases and latent infections. Ophthalmology. 1980;87:1202–1207. [CrossRef] [PubMed]
Metcalf JF Kaufman HE . Herpetic stromal keratitis-evidence for cell-mediated immunopathogenesis. Am J Ophthalmol. 1976;82:827–834. [CrossRef] [PubMed]
Knop N Knop E . Ultrastructural anatomy of CALT follicles in the rabbit reveals characteristics of M cells, germinal centres and high endothelial venules. J Anat. 2005;207:409–426. [CrossRef] [PubMed]
Fukushima A Sumi T Fukuda K . Interleukin 10 and transforming growth factor beta contribute to the development of experimentally induced allergic conjunctivitis in mice during the effector phase. Br J Ophthalmol. 2006;90:1535–1541. [CrossRef] [PubMed]
Gamache DA Dimitrijevich SD Weimer LK . Secretion of proinflammatory cytokines by human conjunctival epithelial cells. Ocul Immunol Inflamm. 1997;5:117–128. [CrossRef] [PubMed]
Wotherspoon AC Hardman-Lea S Isaacson PG . Mucosa-associated lymphoid tissue (MALT) in the human conjunctiva. J Pathol. 1994;174:33–37. [CrossRef] [PubMed]
Knop E Knop N . The role of eye-associated lymphoid tissue in corneal immune protection. J Anat. 2005;206:271–285. [CrossRef] [PubMed]
Knop E Knop N . [Eye-associated lymphoid tissue (EALT) is continuously spread throughout the ocular surface from the lacrimal gland to the lacrimal drainage system]. Ophthalmologe. 2003;100:929–942. [CrossRef] [PubMed]
Liu JT Yue J Ren XB Li H . [Measurement of CD4+CD25+ T cells in breast cancer patients and its significance]. Zhonghua Zhong Liu Za Zhi. 2005;27:423–425. [PubMed]
Medeiros MM Peixoto JR Oliveira AC . Toll-like receptor 4 (TLR4)-dependent proinflammatory and immunomodulatory properties of the glycoinositolphospholipid (GIPL) from Trypanosoma cruzi . J Leukoc Biol. 2007;82:488–496. [CrossRef] [PubMed]
Lewkowicz P Lewkowicz N Sasiak A Tchorzewski H . Lipopolysaccharide-activated CD4+CD25+ T regulatory cells inhibit neutrophil function and promote their apoptosis and death. J Immunol. 2006;177:7155–7163. [CrossRef] [PubMed]
Bell MP Svingen PA Rahman MK Xiong Y Faubion WAJr . FOXP3 regulates TLR10 expression in human T regulatory cells. J Immunol. 2007;179:1893–1900. [CrossRef] [PubMed]
Dai P Jeong SY Yu Y . Modulation of TLR signaling by multiple MyD88-interacting partners including leucine-rich repeat Fli-I-interacting proteins. J Immunol. 2009;182:3450–3460. [CrossRef] [PubMed]
Thebault P Condamine T Heslan M . Role of IFNγ in allograft tolerance mediated by CD4+CD25+ regulatory T cells by induction of IDO in endothelial cells. Am J Transplant. 2007;7:2472–2482. [CrossRef] [PubMed]
Chang JH McCluskey PJ Wakefield D . Toll-like receptors in ocular immunity and the immunopathogenesis of inflammatory eye disease. Br J Ophthalmol. 2006;90:103–108. [CrossRef] [PubMed]
Sha Q Truong-Tran AQ Plitt JR Beck LA Schleimer RP . Activation of airway epithelial cells by toll-like receptor agonists. Am J Respir Cell Mol Biol. 2004;31:358–364. [CrossRef] [PubMed]
Abreu MT Fukata M Arditi M . TLR signaling in the gut in health and disease. J Immunol. 2005;174:4453–4460. [CrossRef] [PubMed]
Kubo T Hatton RD Oliver J Liu X Elson CO Weaver CT . Regulatory T cell suppression and anergy are differentially regulated by proinflammatory cytokines produced by TLR-activated dendritic cells. J Immunol. 2004;173:7249–7258. [CrossRef] [PubMed]
Nakamura K Kitani A Strober W . Cell contact-dependent immunosuppression by CD4(+)CD25(+) regulatory T cells is mediated by cell surface-bound transforming growth factor beta. J Exp Med. 2001;194:629–644. [CrossRef] [PubMed]
Piccirillo CA Letterio JJ Thornton AM . CD4(+)CD25(+) regulatory T cells can mediate suppressor function in the absence of transforming growth factor beta1 production and responsiveness. J Exp Med. 2002;196:237–246. [CrossRef] [PubMed]
Herbst-Kralovetz M Pyles R . Toll-like receptors, innate immunity and HSV pathogenesis. Herpes. 2006;13:37–41. [PubMed]
Krieg AM . Therapeutic potential of Toll-like receptor 9 activation. Nat Rev Drug Discov. 2006;5:471–484. [CrossRef] [PubMed]
Kanzler H Barrat FJ Hessel EM Coffman RL . Therapeutic targeting of innate immunity with Toll-like receptor agonists and antagonists. Nat Med. 2007;13:552–559. [CrossRef] [PubMed]
Conroy H Marshall NA Mills KH . TLR ligand suppression or enhancement of Treg cells? A double-edged sword in immunity to tumours. Oncogene. 2008;27:168–180. [CrossRef] [PubMed]
van Maren WW Jacobs JF de Vries IJ Nierkens S Adema GJ . Toll-like receptor signalling on Tregs: to suppress or not to suppress? Immunology. 2008;124:445–452. [CrossRef] [PubMed]
Liu G Zhao Y . Toll-like receptors and immune regulation: their direct and indirect modulation on regulatory CD4+ CD25+ T cells. Immunology. 2007;122:149–156. [CrossRef] [PubMed]
Zanin-Zhorov A Cahalon L Tal G Margalit R Lider O Cohen IR . Heat shock protein 60 enhances CD4+ CD25+ regulatory T cell function via innate TLR2 signaling. J Clin Invest. 2006;116:2022–2032. [CrossRef] [PubMed]
den Brok MH Sutmuller RP Nierkens S . Synergy between in situ cryoablation and TLR9 stimulation results in a highly effective in vivo dendritic cell vaccine. Cancer Res. 2006;66:7285–7292. [CrossRef] [PubMed]
Sutmuller R Garritsen A Adema GJ . Regulatory T cells and toll-like receptors: regulating the regulators. Ann Rheum Dis. 2007;66 (suppl 3):iii91–iii95. [CrossRef] [PubMed]
Oberg HH Ly TT Ussat S Meyer T Kabelitz D Wesch D . Differential but direct abolishment of human regulatory T cell suppressive capacity by various TLR2 ligands. J Immunol. 2010;184:4733–4740. [CrossRef] [PubMed]
Allan SE Alstad AN Merindol N . Generation of potent and stable human CD4+ T regulatory cells by activation-independent expression of FOXP3. Mol Ther. 2008;16:194–202. [CrossRef] [PubMed]
Allan SE Crome SQ Crellin NK . Activation-induced FOXP3 in human T effector cells does not suppress proliferation or cytokine production. Int Immunol. 2007;19:345–354. [CrossRef] [PubMed]
LaRosa DF Gelman AE Rahman AH Zhang J Turka LA Walsh PT . CpG DNA inhibits CD4+CD25+ Treg suppression through direct MyD88-dependent costimulation of effector CD4+ T cells. Immunol Lett. 2007;108:183–188. [CrossRef] [PubMed]
Chen Q Davidson TS Huter EN Shevach EM . Engagement of TLR2 does not reverse the suppressor function of mouse regulatory T cells, but promotes their survival. J Immunol. 2009;183:4458–4466. [CrossRef] [PubMed]
Kumar A Yu FS . Toll-like receptors and corneal innate immunity. Curr Mol Med. 2006;6:327–337. [CrossRef] [PubMed]
Hajjar AM O'Mahony DS Ozinsky A . Cutting edge: functional interactions between toll-like receptor (TLR) 2 and TLR1 or TLR6 in response to phenol-soluble modulin. J Immunol. 2001;166:15–19. [CrossRef] [PubMed]
Jin X Qin Q Chen W Qu J . Expression of toll-like receptors in the healthy and herpes simplex virus-infected cornea. Cornea. 2007;26:847–852. [CrossRef] [PubMed]
Carpenter S O'Neill LA . Recent insights into the structure of Toll-like receptors and post-translational modifications of their associated signalling proteins. Biochem J. 2009;422:1–10. [CrossRef] [PubMed]
Kurt-Jones EA Belko J Yu C . The role of toll-like receptors in herpes simplex infection in neonates. J Infect Dis. 2005;191:746–748. [CrossRef] [PubMed]
Finberg RW Knipe DM Kurt-Jones EA . Herpes simplex virus and toll-like receptors. Viral Immunol. 2005;18:457–465. [CrossRef] [PubMed]
Figure 1.
 
Intracellular staining of Toll-like receptors in CD4+CD25+ regulatory T cells purified from rabbit conjunctiva. (A) Purified CD4+CD25+ cells (0.66 × 105 per assay) were first surface stained with 1 μL α rabbit CD25-biotin and then intra cellularly stained with 1 μL α human TLR2-PE, TLR3 PE, TLR4-PE, TLR8-PE, TLR9-PE, as shown on each histogram. Dotted line: CD4+CD25+ cells only; solid line: cells stained with anti-human TLR-PE antibody. (B) Isotype control (dotted line) where CD4+CD25+ cells (0.66 × 105) were first stained with 1 μL anti-rabbit CD25-biotin followed by staining with 1 μL mouse IgG1 PE. (C, D) Level of TLRs in CD4+CD25+ and CD4+CD25 cells as a function of MFI. *P < 0.05 when compared with the MFI of TLR2 and TLR9 expression in CD4+CD25+ and CD4+CD25 populations.
Figure 1.
 
Intracellular staining of Toll-like receptors in CD4+CD25+ regulatory T cells purified from rabbit conjunctiva. (A) Purified CD4+CD25+ cells (0.66 × 105 per assay) were first surface stained with 1 μL α rabbit CD25-biotin and then intra cellularly stained with 1 μL α human TLR2-PE, TLR3 PE, TLR4-PE, TLR8-PE, TLR9-PE, as shown on each histogram. Dotted line: CD4+CD25+ cells only; solid line: cells stained with anti-human TLR-PE antibody. (B) Isotype control (dotted line) where CD4+CD25+ cells (0.66 × 105) were first stained with 1 μL anti-rabbit CD25-biotin followed by staining with 1 μL mouse IgG1 PE. (C, D) Level of TLRs in CD4+CD25+ and CD4+CD25 cells as a function of MFI. *P < 0.05 when compared with the MFI of TLR2 and TLR9 expression in CD4+CD25+ and CD4+CD25 populations.
Figure 2.
 
(A) Intracellular staining of Toll-like receptors in CD11b+ APCs isolated from rabbit conjunctiva. CD11b+ cells (0.24 × 106 per assay) were first surface stained with 1 μL anti-rabbit CD11b-FITC antibody and then intracellularly stained with 3 μL anti-human TLR2-PE, TLR3-PE, TLR4-PE, TLR8-PE, TLR9-PE in each set. Dotted line: CD11b+ cells only; solid line: cells stained with anti-human TLR-PE antibody. (B) TLR level in CD11b+ cells as a function of MFI. *P < 0.05 compared with the MFI of TLR2 and TLR9 expression in the CD11b+ populations.
Figure 2.
 
(A) Intracellular staining of Toll-like receptors in CD11b+ APCs isolated from rabbit conjunctiva. CD11b+ cells (0.24 × 106 per assay) were first surface stained with 1 μL anti-rabbit CD11b-FITC antibody and then intracellularly stained with 3 μL anti-human TLR2-PE, TLR3-PE, TLR4-PE, TLR8-PE, TLR9-PE in each set. Dotted line: CD11b+ cells only; solid line: cells stained with anti-human TLR-PE antibody. (B) TLR level in CD11b+ cells as a function of MFI. *P < 0.05 compared with the MFI of TLR2 and TLR9 expression in the CD11b+ populations.
Figure 3.
 
In vitro proliferation of CD4+CD25+ Treg cells purified from rabbit conjunctiva. CFSE-labeled CD4+CD25+ Treg cells were stimulated with 1 μg/mL soluble α-CD3 (A) or α-CD3 + APCs (B) for 6 days at 37°C in a 5% CO2 incubator, in the presence and absence of TLR ligand 2 (LTA) and TLR ligand 9 (CpG2007) at a concentration of 10 μg/mL and 1 μg/mL, respectively. Cells were harvested and stained with anti-rabbit CD4-PE or anti-rabbit CD25-PE and analyzed by flow cytometry. Values in each bar indicate the average ± SD of two wells. Each well has 5 × 104 CFSE-labeled CD4+CD25+ Treg cells and with and without 1 × 105 APCs (mitomycin C treated), as designated in the figure.
Figure 3.
 
In vitro proliferation of CD4+CD25+ Treg cells purified from rabbit conjunctiva. CFSE-labeled CD4+CD25+ Treg cells were stimulated with 1 μg/mL soluble α-CD3 (A) or α-CD3 + APCs (B) for 6 days at 37°C in a 5% CO2 incubator, in the presence and absence of TLR ligand 2 (LTA) and TLR ligand 9 (CpG2007) at a concentration of 10 μg/mL and 1 μg/mL, respectively. Cells were harvested and stained with anti-rabbit CD4-PE or anti-rabbit CD25-PE and analyzed by flow cytometry. Values in each bar indicate the average ± SD of two wells. Each well has 5 × 104 CFSE-labeled CD4+CD25+ Treg cells and with and without 1 × 105 APCs (mitomycin C treated), as designated in the figure.
Figure 4.
 
Quantification of INF-γ (A, D), IL-10 (B, E), and TGF-β (C, F) expression in rabbit conjunctiva-purified CD4+CD25+ cells using real-time PCR. One million nTreg cells were plated in a six-well tissue culture plate and were pretreated with LTA (10 μg/mL; AC) or CpG (1 μg/mL; DF) for 12 hours, followed by stimulation for another 12 hours in the presence of anti-CD3 (1 μg/mL) and IL-2 (20 U/mL) at 37°C. RNA was isolated from harvested cells and was quantified. An equal amount of RNA was reversed transcribed. cDNA was amplified using real time PCR and specific primers for rabbit INF-γ, IL-10, and TGF-β. GAPDH was used as a housekeeping gene. RNA quantification was calculated using the comparative Ct method, also known as the 2-ΔΔCt method, where ΔΔCt = ΔCt sample − ΔCt reference (none). Here, ΔCT sample is the Ct value for any sample normalized to the endogenous housekeeping gene, and ΔCt reference (none) is the Ct value for the calibrator also normalized to the endogenous housekeeping gene.
Figure 4.
 
Quantification of INF-γ (A, D), IL-10 (B, E), and TGF-β (C, F) expression in rabbit conjunctiva-purified CD4+CD25+ cells using real-time PCR. One million nTreg cells were plated in a six-well tissue culture plate and were pretreated with LTA (10 μg/mL; AC) or CpG (1 μg/mL; DF) for 12 hours, followed by stimulation for another 12 hours in the presence of anti-CD3 (1 μg/mL) and IL-2 (20 U/mL) at 37°C. RNA was isolated from harvested cells and was quantified. An equal amount of RNA was reversed transcribed. cDNA was amplified using real time PCR and specific primers for rabbit INF-γ, IL-10, and TGF-β. GAPDH was used as a housekeeping gene. RNA quantification was calculated using the comparative Ct method, also known as the 2-ΔΔCt method, where ΔΔCt = ΔCt sample − ΔCt reference (none). Here, ΔCT sample is the Ct value for any sample normalized to the endogenous housekeeping gene, and ΔCt reference (none) is the Ct value for the calibrator also normalized to the endogenous housekeeping gene.
Figure 5.
 
In vitro suppression of CD+CD25 effector cells by CD4+CD25+ nTreg. Conjunctiva-purified CD+CD25 effector cells (5 × 104 cells) were labeled with CFSE and stimulated with soluble α human CD3 (1 μg/mL) and mitomycin C (50 μg/mL)–treated autologous APCs (1 × 105 cells) for 5 days at 37°C. For the suppression assay, CFSE-labeled CD4+CD25 effector cells (5 × 104 cells) were mixed with nonlabeled CD4+CD25+ cells (10 × 104 cells) in culture medium containing soluble α human CD3 (1 μg/mL) and autologous APCs (1 × 105 cells) and were incubated in the presence and absence of LTA (10 μg/mL) and CpG2007 (1 μg/mL) for 5 days at 37°C. Cells were harvested and stained with α rabbit CD4-PE and analyzed by flow cytometry. (A, dotted line) Absolute number of CD4+CD25 effector cells in the absence of anti-human CD3 and autologous APCs. (B) Percentage of Teff cells calculated from (A). Values in each bar indicate the average ± SD of two wells. (C) Histogram that represents nonstimulated CFSE-labeled CD4+CD25 Teff cells (dashed lines) overlaid with histograms (bold lines) that represent CFSE-labeled CD4+CD25 Teff cells stimulated with (a) an anti-CD3+ mAb alone or with (b) an anti-CD3+ mAb in the presence of untreated CD4+CD25+ Treg cells, (c) an anti-CD3+ mAb in the presence of LTA-treated CD4+CD25+ Treg cells, or (d) an anti-CD3+ mAb in the presence of CpG-treated CD4+CD25+ Treg cells.
Figure 5.
 
In vitro suppression of CD+CD25 effector cells by CD4+CD25+ nTreg. Conjunctiva-purified CD+CD25 effector cells (5 × 104 cells) were labeled with CFSE and stimulated with soluble α human CD3 (1 μg/mL) and mitomycin C (50 μg/mL)–treated autologous APCs (1 × 105 cells) for 5 days at 37°C. For the suppression assay, CFSE-labeled CD4+CD25 effector cells (5 × 104 cells) were mixed with nonlabeled CD4+CD25+ cells (10 × 104 cells) in culture medium containing soluble α human CD3 (1 μg/mL) and autologous APCs (1 × 105 cells) and were incubated in the presence and absence of LTA (10 μg/mL) and CpG2007 (1 μg/mL) for 5 days at 37°C. Cells were harvested and stained with α rabbit CD4-PE and analyzed by flow cytometry. (A, dotted line) Absolute number of CD4+CD25 effector cells in the absence of anti-human CD3 and autologous APCs. (B) Percentage of Teff cells calculated from (A). Values in each bar indicate the average ± SD of two wells. (C) Histogram that represents nonstimulated CFSE-labeled CD4+CD25 Teff cells (dashed lines) overlaid with histograms (bold lines) that represent CFSE-labeled CD4+CD25 Teff cells stimulated with (a) an anti-CD3+ mAb alone or with (b) an anti-CD3+ mAb in the presence of untreated CD4+CD25+ Treg cells, (c) an anti-CD3+ mAb in the presence of LTA-treated CD4+CD25+ Treg cells, or (d) an anti-CD3+ mAb in the presence of CpG-treated CD4+CD25+ Treg cells.
Figure 6.
 
Six rabbits (two per group) were immunized ocularly three times at an interval of 14 days with a mixture of four HSV-gD peptide (gD144–179, gD287–317, gD49–82, and gD332–358) mixed with either CpG2007 (group 1) or LTA (group 2) as a topical ocular mucosal immunoadjuvants. The control group received saline alone, LTA alone, or CpG2007 alone (mock, group 3). Ten days after the third immunization, rabbits were euthanatized, upper and lower conjunctivas were harvested from each rabbit, and lymphocytes were isolated. Lymphocytes were labeled with CFSE and stimulated with HSV-gD peptide (gD144–179, gD287–317, gD49–82, or gD332–358)–pulsed autologous APCs for 5 days at 37°C in a CO2 incubator. Cells were harvested and stained with anti-rabbit CD4-PE, and their proliferation was analyzed by flow cytometry. (A) Absolute number of CFSE-labeled proliferating CD4+ cells under the marked conditions. (B) Dot plot representation with the percentage of CFSE-labeled proliferating CD4+ cells.
Figure 6.
 
Six rabbits (two per group) were immunized ocularly three times at an interval of 14 days with a mixture of four HSV-gD peptide (gD144–179, gD287–317, gD49–82, and gD332–358) mixed with either CpG2007 (group 1) or LTA (group 2) as a topical ocular mucosal immunoadjuvants. The control group received saline alone, LTA alone, or CpG2007 alone (mock, group 3). Ten days after the third immunization, rabbits were euthanatized, upper and lower conjunctivas were harvested from each rabbit, and lymphocytes were isolated. Lymphocytes were labeled with CFSE and stimulated with HSV-gD peptide (gD144–179, gD287–317, gD49–82, or gD332–358)–pulsed autologous APCs for 5 days at 37°C in a CO2 incubator. Cells were harvested and stained with anti-rabbit CD4-PE, and their proliferation was analyzed by flow cytometry. (A) Absolute number of CFSE-labeled proliferating CD4+ cells under the marked conditions. (B) Dot plot representation with the percentage of CFSE-labeled proliferating CD4+ cells.
Figure 7.
 
Foxp3 expression on rabbit conjunctival Treg by TLR2 and TLR9 ligands. Rabbit conjunctiva–purified CD4+CD25+ Treg cells (0.5 × 106 cells) were either left untreated or stimulated overnight with anti-human CD3 (1 μg/mL) ± treatment with LTA (10 μg/mL), CpG (1 μg/mL), or Pam3CSK4 (50 ng/mL). TLR ligand treatments and anti-CD3 stimulations were performed together in a 90-well, flat-bottomed tissue culture plate at 37°C/5% CO2 incubator for overnight. (A, B), Cells were harvested and surface stained with anti-rabbit CD4-FITC (10 μL/assay) followed by intracellular staining with anti-human Foxp3-PE (20 μL/assay) and were analyzed by FACS. (C) Cells from each well (identical to those described) were lysed with 100-μL lysis buffer (RIPA with PI cocktail). Equal volumes (20 μL) of denatured cell lysates containing ∼1 × 105 cells were loaded in each lane. Details of the Western blot procedure have been previously described. 1 (A, dot plot) MFI of CD4+/Foxp3-positive cells for rabbit conjunctiva-purified Treg cells (top) and human PBMC-purified Treg cells (bottom). Human PBMC-purified Treg cells were used as a positive control in this assay. (B, overlapped histograms) Expression of Foxp3 in Treg by FACS. (C) Foxp3 protein in rabbit conjunctiva-purified CD4+CD25+ Treg cells detected by Western blot analysis.
Figure 7.
 
Foxp3 expression on rabbit conjunctival Treg by TLR2 and TLR9 ligands. Rabbit conjunctiva–purified CD4+CD25+ Treg cells (0.5 × 106 cells) were either left untreated or stimulated overnight with anti-human CD3 (1 μg/mL) ± treatment with LTA (10 μg/mL), CpG (1 μg/mL), or Pam3CSK4 (50 ng/mL). TLR ligand treatments and anti-CD3 stimulations were performed together in a 90-well, flat-bottomed tissue culture plate at 37°C/5% CO2 incubator for overnight. (A, B), Cells were harvested and surface stained with anti-rabbit CD4-FITC (10 μL/assay) followed by intracellular staining with anti-human Foxp3-PE (20 μL/assay) and were analyzed by FACS. (C) Cells from each well (identical to those described) were lysed with 100-μL lysis buffer (RIPA with PI cocktail). Equal volumes (20 μL) of denatured cell lysates containing ∼1 × 105 cells were loaded in each lane. Details of the Western blot procedure have been previously described. 1 (A, dot plot) MFI of CD4+/Foxp3-positive cells for rabbit conjunctiva-purified Treg cells (top) and human PBMC-purified Treg cells (bottom). Human PBMC-purified Treg cells were used as a positive control in this assay. (B, overlapped histograms) Expression of Foxp3 in Treg by FACS. (C) Foxp3 protein in rabbit conjunctiva-purified CD4+CD25+ Treg cells detected by Western blot analysis.
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