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Original Article |

Toll-like Receptors in Regulatory T Cells of Patients With Head and Neck Cancer FREE

Clarissa Anni Wild, PhD; Sven Brandau, PhD; Monika Lindemann, PhD; Ramin Lotfi, MD; Thomas K. Hoffmann, MD; Stephan Lang, MD; Christoph Bergmann, MD
Arch Otolaryngol Head Neck Surg. 2010;136(12):1253-1259. doi:10.1001/archoto.2010.195.
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Published online

Objective  To investigate the role of Toll-like receptor (TLR) signaling and T-regulatory (T-reg) cells in patients with head and neck squamous cell carcinoma (HNSCC).

Design  Multicolor flow cytometry was used to study the frequency and phenotype of CD4+CD25+CD127 T-reg cells and CD4+CD25CD127+ T-effector (T-eff) cells in peripheral blood mononuclear cells (PBMCs).

Setting  All patients were seen at the outpatient clinic at the Department of Otorhinolaryngology at the University of Duisburg–Essen from March 1, 2009, through December 31, 2009.

Patients  Eleven patients with HNSCC and 10 healthy donors (HDs) were studied. T-reg and T-eff cells were isolated from PBMCs using a magnetic bead–activated cell-sorting technique.

Main Outcome Measures  Proliferation of T-eff cells and suppressor activity of T-reg cells were assessed in functional assays after preincubation with the TLR4 ligand heat shock protein 60 or lipopolysaccharide in the presence or absence of neutralizing antibody against TLR4.

Results  Frequency of T-reg cells in PBMCs was strongly increased in patients with HNSCC vs HDs. Isolation of T-reg cells from PBMCs of patients with HNSCC showed a significantly higher expression of TLR4, TLR6, TLR9, and TLR10 compared with HDs, whereas TLR2 was not detectable. After incubation with heat shock protein 60 or lipopolysaccharide, the suppressive function of T-reg cells was significantly increased (1.14- and 1.44-fold, respectively), whereas the proliferation capacity of T-eff cells remained unchanged. This effect was reversed after TLR4 inhibition on T-reg cells.

Conclusion  The TLR ligation on T-reg cells may contribute to tumor-mediated immune suppression by enhancing their suppressive activity.

Figures in this Article

Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignant neoplasm with a 5-year patient survival rate of less than 50%, which has remained unchanged for the past 30 years despite advances in diagnostics and treatment.1 Although the origin of the process of tumorigenesis is clearly linked to environmental carcinogens (eg, tobacco and alcohol) ensuing in a plethora of epigenetic events, which govern the development of HNSCC, its progression is strongly influenced by the host immune system and results in emergence of a tumor that is resistant to immune effector cells.2,3

A variety of strategies to evade immune detection and elimination have evolved in HNSCC. A mechanism of immune suppression that has recently been emphasized involves T-regulatory (T-reg) cells. Although T-reg cells maintain tolerance against their own antigens and prevent development of autoimmunity in healthy individuals, tumor cells take advantage of T-reg cells to protect themselves by suppressing antitumor immune responses.4

T-reg cells are currently classified into 2 main subsets according to their origin and suppressive activity.5 Natural T-reg cells originate in the thymus and have been described to suppress T-effector (T-eff) cell responses in a contact-dependent and cytokine-independent manner. They are mainly characterized by expression of interleukin (IL) 2 receptor α (CD25) and the transcription factor forkhead box P3 (FoxP3). Adaptive or induced T-reg cells are antigen-specific suppressor cells that are activated during inflammatory processes in peripheral tissues and mediate their suppressive activity through secretion of IL-10 and transforming growth factor β, respectively.6

One of us and others79 have reported on the presence of natural T-reg cells and induced T-reg cells in patients with HNSCC. Those works have demonstrated that the presence of either T-reg subset was strongly associated with tumor progression and that both subsets showed enhanced suppressive activity compared with T-reg cells in healthy individuals.

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns or damage-associated molecular patterns and have recently emerged as critical key mediators of immune functions. Several immune cells from the innate system10 and the more recently described adaptive immune system11 express a variety of TLRs. Activation of immune cells through TLRs results in enhanced immune responses against exogenous (pathogen-associated molecular patterns, ie, lipopolysaccharide [LPS]) or endogenous (damage-associated molecular patterns, ie, heat shock protein 60 [HSP60]) molecules during immune defense. Recently, a different study12 has shown that tumor cells are able to modulate the expression and function of TLR proteins in different kinds of immune cells, resulting in impaired immune function.

The aim of the present study was to investigate the role of TLR signaling and T-reg cells in patients with HNSCC. The expression of TLRs on T-reg and T-eff cells in the peripheral blood of patients with HNSCC was evaluated and the functional effects of TLR4 ligands on T-reg and T-eff cell activity were analyzed. The rationale for the study was based on the hypothesis that tumor cells recruit T-reg cells via secreting soluble factors that activate T-reg cells through TLR ligation, which in turn act against an antitumor immune response. Our results suggest that activation of T-reg cells through TLR4 ligation in patients with HNSCC cells may contribute to tumor-mediated immune suppression.

REAGENTS AND ANTIBODIES

Biocoll separating solution was used for density centrifugation of peripheral blood and fetal calf serum was inactivated by heating for 30 minutes to 56°C (Biochrom AG, Berlin, Germany). We also used RPMI 1640 culture medium, L-glutamine, streptomycin, and penicillin (Invitrogen, Karlsruhe, Germany). Complete medium (Rxx10) consisting of RPMI 1640 supplemented with 10% (vol/vol) fetal calf serum, 100-IU/mL penicillin, 100-μg/mL streptomycin, and L-glutamine (2 mmol/L) was used for cell culture assay. The following monoclonal antibodies (mAbs) were used for flow cytometry: anti–CD4-perdinin chlorophyll, anti–CD25-allophycocyanin, anti–TLR2-Alexa Fluor 488, and 7–amino actinomycin D (7-AAD) (BD Biosciences, Heidelberg, Germany); anti–CD127-phycoerythrin (PE)-Cy7, anti–FoxP3-fluorescein isothiocyanate (FITC), and functional grade purified anti–TLR4 (eBioscience Corp, Frankfurt am Main, Germany); and anti–TLR6-PE, anti–TLR9-PE (Imgenex Corp, San Diego, California), anti–TLR4-PE (Abcam, Cambridge, England) and anti–TLR10-FITC (Santa Cruz Biotechnology Inc, Heidelberg, Germany).

COLLECTION OF PERIPHERAL BLOOD MONONUCLEAR CELLS FROM PATIENTS WITH HNSCC AND HEALTHY DONORS

Blood samples were obtained from 11 patients with HNSCC and 10 healthy donors (HDs) as control individuals. Peripheral venous blood (50-150 mL) was drawn into heparinized tubes, diluted (1:1, vol/vol) with phosphate-buffered saline (PBS), and subjected to density gradient centrifugation (room temperature, 300 g, 30 minutes). Peripheral blood mononuclear cells (PBMCs) were recovered, washed with PBS, counted (CasyCounter; Innovatis-Roche, Bielefeld, Germany), and immediately used for experiments.

The study was approved by the institutional review board of the University of Duisburg–Essen, and all participants signed an informed consent form. All patients were seen at the outpatient clinic at the Department of Otorhinolaryngology at the University of Duisburg–Essen from March 1, 2009, through December 31, 2009. The patient cohort included men and women with a mean age of 68 years (range, 55-82 years). The HD group was sex matched and included 6 men and 4 women with a mean age of 36 years (range, 22-46 years), all of whom were nonsmokers. All patients had untreated primary tumors. The age, sex, and clinicopathologic characteristics of the patients are listed in the Table.

Table Graphic Jump LocationTable. Clinicopathologic Characteristics of Study Patients With Head and Neck Squamous Cell Carcinoma
ISOLATION OF CD4+CD25+ T-REG CELLS

For magnetic bead purification, a CD4+CD25+ T-reg isolation kit was used according to the manufacturer's protocol (Miltenyi Biotec, Mönchengladbach, Germany). In brief, all non-CD4 cells were depleted over a magnetic cell separater (MACS) LD column (Miltenyi Biotech) after incubation with a cocktail of biotin-conjugated mAbs against CD8, CD14, CD16, CD19, CD36, CD56, CD123, CD127, T-cell receptor γ/δ, and CD235a. The unlabeled CD4+ T cells were incubated with CD25 microbeads and subsequently separated over 2 MACS MS columns (Miltenyi Biotech) into CD4+CD25+ T-reg cells and CD4+CD25 T-eff cells. Purity of cell subsets was routinely tested and ranged from 90% to 97%.

FLOW CYTOMETRY

Freshly isolated PBMCs were stained for TLR expression. Therefore, cells were incubated in sodium azide–PBS containing 3% human serum with the optimal dilution of each mAb for 25 minutes at 4°C in the dark. The following antibodies were used for surface staining: anti–CD4-perdinin chlorophyll (TH1 cells), anti–CD25-allophycocyanin (IL-2 receptor α), anti–CD127-PE-Cy7 (IL-7 receptor α), anti–TLR2-Alexa Fluor 488, anti–TLR4-PE, anti–TLR6-PE, and anti–TLR10-FITC. Cells were subsequently washed and fixed with 2% (vol/vol) paraformaldehyde or proceeded to intracellular staining (FoxP3, TLR9) (eBioscience Corp). For this purpose, cells were incubated with fixation and permeabilization working solution for 30 minutes at 4°C in the dark, washed twice with permeabilization buffer, and incubated with mAbs diluted in the same buffer for 25 minutes at 4°C in the dark. All mAbs used were pretitrated on fresh PBMCs to determine their optimal working dilutions. Respective isotype controls were used in all experiments (all corresponding isotypes were purchased from BD Biosciences except for ratIgG2a-FITC, which was obtained from Dianova Inc, Hamburg, Germany). After staining, cells were immediately subjected to acquisition in a flow cytometer (FACSCanto II; BD Biosciences). The collected data were analyzed with Diva6 software (Becton Dickinson, Heidelberg, Germany).

INCORPORATION ASSAY

The T-eff cells were preincubated for 2 hours with LPS (Microbiology and Infection Biology, Division of Biophysics, Research Center Borstel, Borstel, Germany) at different concentrations and extensively washed before adding CD2/CD3/CD28 MACS bead particles (MACSiBead; Miltenyi Biotec), according to the manufacturer's instructions. Assays were conducted in quadruplicate on a 96-microtiter well plate (Greiner Bio-One, Frickenhausen, Germany) in a total volume of 0.2 mL for 3 days. For the last 16 hours cells were labeled with 1 μCi thymidine labeled with tritium per well (TRA.120; specific activity, 5 Ci/mmol; Amersham, Buckinghamshire, England). Cells were then harvested (Micro96 Harvester; Skatron Instruments, Transby, Norway) onto glass fiber filtermates (PerkinElmer, Turku, Finland), and the incorporated radioactivity was quantified by liquid scintillation counting (1450 Microbeta Trilux; PerkinElmer).

CARBOXYFLUORESCEIN DIACETATE SUCCINIMIDYL ESTER–BASED SUPPRESSION ASSAY AND FLOW CYTOMETRY–BASED CYTOTOXICITY ASSAY

Responder CD4+CD25 T cells were MACS isolated from PBMCs, stained with 5-μM carboxyfluorescein diacetate succinimidyl ester (CFSE), and cocultured with autologous suppressor cells. For that purpose, T-reg cells were preincubated with 100-ng/mL LPS or 10-ng/mL HSP60 (Lionex GmbH, Braunschweig, Germany) in Rxx10 for 2 hours. To analyze the role of TLR4 signaling, neutralizing mAb (20 μg/mL) and corresponding monoclonal IgG2a isotype control, which were previously titrated, were added to T-reg cells in some experiments 25 minutes before LPS stimulation. Afterward, cells were extensively washed with complete medium and activated overnight with CD2/CD3/CD28 MACS bead particles. On the following day, 5 × 104 autologous CFSE-labeled T-eff cells were seeded into 96 round-bottom plates. T-reg cells were again washed and added at various ratios (ratio of responder to suppressor cells, 1:1, 3:1, 10:1) to the wells supplemented with CD2/CD3/CD28 MACS bead particles as recommended in the manufacturer's instructions. This assay was performed in triplicate. Cocultures were incubated for 5 days at 37°C in an atmosphere of 5% carbon dioxide in air.

To assess cytotoxic lysis of T-eff cells induced by T-reg cells, a flow cytometry–based cytotoxicity assay was performed as previously described.13 Briefly, directly before analysis, cells were stained in PBS with the vital dye 7-AAD for 15 minutes at room temperature. The 7-AAD intercalates into double-stranded DNA and emits red fluorescent light. The 7-AAD cannot pass through intact cell membrane and thus is a helpful surrogate parameter for late cell death or apoptosis. Cells were resuspended in 200 μL of PBS and were immediately subjected to acquisition in a flow cytometer (FACSCanto II). Flow cytometry analysis and CFSE data analysis were performed using Diva6 software.

STATISTICAL ANALYSIS

All data are presented as mean (SD) of at least 3 experiments. Data were analyzed using the t test and Mann-Whitney rank sum test for comparisons across outcome groups (HDs vs patients with HNSCC). Data are presented in box plots showing the mean, median, and interquartile range. A significance level of .05 was assumed.

FREQUENCY OF CD4+CD25+CD127 T-REG CELLS IN PATIENTS WITH HNSCC

The PBMCs obtained from the 11 patients with HNSCC and the 10 HDs were first investigated for the frequency of CD4+CD25+CD127 T-reg cells. For this purpose, 6-color flow cytometry was used. A previously reported gating strategy to identify highly functional T-reg cells14 was used to assess the frequency of T-reg cells. Briefly, after gating on the lymphocyte fraction as to their front scatter and side scatter properties, CD4+ cells were gated and followed by T-reg identification using IL-2 receptor α chain (CD25) and IL-7 receptor α chain (CD127) (Figure 1A). The percentages of circulating CD4+CD25+CD127 T-reg cells were significantly increased in patients with HNSCC (7.7% [1.6%]) compared with HDs (4.0% [1.1%]; P < .001) (Figure 1B). FoxP3 as another eminent functional marker for T-reg cell identification was also assessed by flow cytometry.15 On average, 71.3% (7.4%) of CD4+CD25+CD127 T-reg cells from HDs showed positive results for FoxP3, whereas 86.3% (2.1%) showed positive results in the HNSCC cells (data not shown).

Place holder to copy figure label and caption
Figure 1.

Frequency of T-regulatory (T-reg) cells in patients with head and neck squamous cell carcinoma (HNSCC). A, Gating strategy to analyze T-reg and T-effector (T-eff) cells in peripheral blood mononuclear cells of patients with HNSCC or healthy donors (HDs): first, gate was set on lymphocytes according to their forward or sideward scatter properties. Within the CD4+ T-cell population, T-reg cells were identified as CD25+CD127, whereas T-eff cells are represented as CD25CD127+ cells. B, Percentages of CD4+CD25+CD127 T-reg cells of 11 patients with HNSCC and 10 HDs are shown. Data are mean (SD) as determined by flow cytometry.

Graphic Jump Location
INCREASED TLR EXPRESSION ON CD4+CD25+CD127 T-REG CELLS IN PATIENTS WITH HNSCC

To identify the potential role of TLR on T-reg cells in patients with HNSCC, we investigated the phenotypic TLR profile on CD4+CD25+CD127 T-reg cells using flow cytometry. Except for TLR2, all TLRs tested were detected on peripheral T-reg cells. Expression levels were quantified by determining the median fluorescence intensity, defined as the difference between the staining intensity of the isotype control and the TLR-specific antibody. The constitutive expression level of TLR4, TLR6, TLR9, and TLR10 was significantly increased (P < .001) on the T-reg cells of patients with HNSCC compared with those of HDs: TLR4, 163.3 vs 803.0; TLR6, 160.9 vs 386.4; TLR9, 305.0 vs 1001.9; and TLR10, 13.3 vs 69.9. Box plots in Figure 2A summarize the mean, median, and interquartile range of the TLR expression on T-reg cells. Representative flow cytometry histograms show the difference in the expression levels of TLR4, TLR6, TLR9, and TLR10 on T-reg cells of HDs vs those of patients with HNSCC (Figure 2B). Flow cytometry analysis of TLR on T-eff cells revealed similar expression levels (data not shown).

Place holder to copy figure label and caption
Figure 2.

Constitutive expression of Toll-like receptor (TLR) on T-regulatory (T-reg) cells. A, Expression of TLR4, TLR6, TLR9, and TLR10 on T-reg cells isolated from peripheral blood mononuclear cells (PBMCs) of 11 patients with head and neck squamous cell carcinoma (HNSCC) and 10 healthy donors (HDs) as obtained by flow cytometry is shown as mean fluorescent intensity defined as the difference between the staining intensity of the isotype control and the TLR-specific antibody (ΔMFI). Box plots present the mean, median, and interquartile range. B, Histograms illustrate the difference in the expression level of TLR4, TLR6, TLR8, and TLR10 on T-reg cells of patients with HNSCC vs HDs. Flow cytometry histograms shown are representative for all stained PBMCs. Anti–TLR4-FITC-A indicates antibody against TLR4 receptor, conjugated to the FITC fluorochrome; anti–TLR6-FITC-A, antibody against TLR6 receptor, conjugated to the FITC fluorochrome; anti–TLR10-FITC-A, antibody against TLR10 receptor, conjugated to the FITC fluorochrome; and TLR9-PE-A, antibody against TLR9 receptor, conjugated to the PE fluorochrome.

Graphic Jump Location
EFFECT OF LPS ON T-EFF CELL PROLIFERATION

To assess the role of TLR on T-eff and T-reg cells, we chose LPS as a paradigmatic TLR4 ligand. MACS-isolated CD4+CD25 T-eff cells were preincubated for 2 hours with 0.01-, 0.1-, or 1-μg/mL LPS at 37°C in an atmosphere of 5% carbon dioxide in air. Cells were washed and allowed to proliferate for 3 days on anti-CD2/CD3/CD28 activation. This short exposure to LPS did not alter proliferation capacity of T-eff cells in either an enhancing or an inhibiting manner. Counts per minute of proliferated T-eff cells ranged from 50 000 to 55 000, as shown in Figure 3.

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Figure 3.

Proliferative response of T-effector (T-eff) cells to lipopolysaccharide (LPS) stimulation. T-eff cells were preincubated for 2 hours with LPS at different concentrations as indicated on the x-axis. Cells were washed and activated with αCD2/CD3/CD28 beads. Counts per minute (CPMs) were measured after 72 hours. Thymidine labeled with tritium was added for the last 16 hours. Data shown are mean (SD) CPMs derived from experiments with cells of 4 different donors. Plus signs indicate positive; minus signs, negative.

Graphic Jump Location
EFFECT OF LPS ON THE SUPPRESSIVE FUNCTION OF T-REG CELLS

Next, we sought to determine whether LPS as an exogenous ligand or HSP60 as an endogenous ligand modulates the suppressive function of T-reg cells. In previous experiments, different LPS or HSP60 concentrations ranging from 0.001 to 1 μg/mL were tested in preliminary runs of CFSE suppression assays. A concentration of LPS at 0.1 μg/mL and a concentration of HSP60 at 0.01 μg/mL showed the strongest results. Therefore, MACS-isolated CD4+CD25+ T-reg cells were preincubated for 2 hours with 0.1-μg/mL LPS or 0.01-μg/mL HSP60 and activated overnight with CD2/CD3/CD28 MACS bead particles. Next, T-reg cells were washed and added to autologous T-eff cells, which were labeled with CFSE. Cocultures were activated in presence of CD2/CD3/CD28 MACS bead particles for 5 days. In these runs, T-reg cells, which were preactivated with HSP60, exhibited a 1.14-fold (±0.17-fold) increase of suppressor activity compared with untreated T-reg cells. Furthermore, T-reg cells pretreated with LPS showed a 1.44-fold (±0.19-fold) increase of suppressing T-cell proliferation compared with untreated T-reg cells (Figure 4).

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Figure 4.

Suppressive capacity of T-regulatory (T-reg) cells on stimulation with heat shock protein 60 (HSP60) or lipopolysaccharide (LPS); P = .04 for all. A, Magnetic associated cell sorter–sorted CD4+CD25+ T-reg cells were preincubated for 2 hours with 0.1-μg/mL LPS or 0.01-μg/mL HSP60, washed extensively, and activated overnight with αCD2/CD3/CD28 beads. On the following day, autologous carboxyfluorescein diacetate succinimidyl ester (CFSE)–labeled CD4+CD25 T-eff (responder) and T-reg (suppressor) cells were seeded at different ratios in U-bottom 96-well plates. Cells were cocultured in the presence of αCD2/CD3/CD28 beads. On day 5, proliferation of responder cells was analyzed using mean fluorescent intensity of CFSE. The data are mean (SD) x-fold suppression compared with untreated T-reg cells in cocultures with responder cells as derived from experiments with cells of 4 different donors. Plus signs indicate positive; minus signs, negative.

Graphic Jump Location

To assess TLR4-dependent suppressor activity of T-reg cells on inhibition of T-cell proliferation, flow cytometry was used with gates set on CFSE-positive cells. At the responder to suppressor cell ratio of 1:1, the mean percentage of proliferation was 28.3% (4.55%) for autologous CD4+CD25 T-eff cells compared with proliferation of T-eff cells alone. When T-reg cells were preincubated with LPS, proliferation of autologous T-eff cells was further significantly impaired with a mean of 22.3% (7.2%) (Figure 5A). Inhibition of TLR4 using a functional-grade neutralizing antibody against human TLR4 epitopes resulted in abrogation of TLR4-specific enhancement of T-reg cell suppressor activity. Figure 5B (left column) shows a representative example of 4 experiments.

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Figure 5.

Suppressive activity of T-regulatory (T-reg) cells is mediated through Toll-like receptor 4 (TLR4) but does not confer cytotoxic effects on activated T cells;  = .049 for all. A, Proliferation of T-effector (T-eff) cells was evaluated in carboxyfluorescein diacetate succinimidyl ester (CFSE) suppression cultures. The T-eff cells were labeled with CFSE and activated with αCD2/CD3/CD28 beads in cocultures with T-reg cells for 5 days as described in the “Materials and Methods” section. The T-reg cells were pretreated with lipopolysaccharide (LPS) or with neutralizing antibody against TLR4 as indicated below the graph. The data are mean percentage of proliferation derived from experiments with cells of 4 healthy donors. B, CFSE histograms show 1 representative example of 4 independent experiments. Percentages indicate proliferation of CFSE-positive responder cells (left column) Flow cytometry–based cytotoxicity assay simultaneously measures induction of apoptosis and proliferation of CD4+CD25 T-effector (T-eff) cell proliferation and suppression mediated by T-reg cells. Percentages indicate 7–amino actinomycin D (7-AAD)–positive T-eff cells, which represent cells undergoing apoptosis (right column). Plus signs indicate positive; minus signs, negative.

Graphic Jump Location

Furthermore, to determine the effect of T-reg cell suppression on T-eff cells, we used flow cytometry–based cytotoxicity assay. Flow cytometry–based cytotoxicity assay discriminates between CFSE-labeled, 7-AAD–positive (dead) responder cells and unlabeled, 7-AAD–negative (live) responder cells and simultaneously measures the extent of proliferation inhibition (CFSE dye dilution) in responder cell populations. In a coculture (responder cell plus T-reg cells) established with HD cells, T-reg cells induced little apoptosis (5.9% of 7-AAD–positive responder cells), and they mediated moderate suppression (29.2%) of responder cell proliferation. When pretreated with LPS, proliferation of responder cells was 19.8% compared with responder cell proliferation alone, whereas the proportion of dead responder cells remained steady at 5.3%. The LPS-treated T-reg cells in the presence of neutralizing antibody against TLR4 mediated an inhibition of proliferation (28.2%) and a percentage of apoptotic cells (6.1%) comparable with untreated T-reg cells (Figure 5B). These results suggest that suppressor activity of T-reg cells is in part dependent on TLR4-mediated signaling but does not confer cytotoxic effects on proliferating T-eff cells.

It is now well recognized that TLRs play a pivotal role in the activation of innate immunity against invading pathogens, induction of cytokine production, and development of adaptive immune responses.10 In contrast with the protective role of TLRs against pathogen infections, this study suggests that TLRs might play an oppositional role biologically in the context of tumor progression in patients with cancer.

Consistent with the findings of previous studies by some of us,8,16 we now confirm that the frequency of T-reg cells with a high suppressor phenotype is significantly elevated in patients with HNSCC. Previously, some of us had further reported that CD4+CD25highFoxP3+ T-reg cells in patients with HNSCC suppress proliferation of CD4+ T cells by a granzyme/perforin-dependent mechanisms and kill CD8+ T-eff cells by inducing Fas-mediated apoptosis.13 In aggregate, this emphasizes the contribution of T-reg cells to a highly immunosuppressive tumor microenvironment. Within this microenvironment, orchestration of the soluble factors secreted by the tumor cells or altered stromal cells may not only inhibit immune cell function directly or by induction of apoptosis of T-eff cells17 but also might be perceived by TLRs expressed on T-reg cells. Furthermore, it has been demonstrated that HNSCC cells themselves express TLR4 among other TLRs, and LPS promotes tumor cell proliferation.18 This would further contribute to the induction of an immunosuppressive environment, which, at least in part, may be used by T-reg cells recruited by the tumor.

For the first time, to our knowledge, we present evidence of the expression of TLRs on T-reg cells in patients with HNSCC. This allows a presumption that TLR signaling on T-reg cells in patients with cancer might play a significant role and it is worthwhile to further elucidate this idea by future studies.

Recent findings indicate that TLRs modulate T-reg cell activity directly in infection biology. Netea et al19 demonstrated that TLRs directly promote the suppressive activity of T-reg cells in fungal infections. Using TLR4 as an example, we demonstrated that its exogenous ligand, LPS, or its endogenous ligand, HSP60, augments suppressive function of T-reg cells. This finding agrees with that of the study by Caramalho et al,20 who reported that LPS treatment of T-reg cells increases their suppressor efficiency by 10-fold. In contrast, we observed that LPS has no effect on T-eff cell proliferation.

Several strategies have been proposed to combine T-reg cell neutralization with conventional antitumor therapies.21,22 Among those, linking TLR signaling to the functional control of T-reg cells may offer new opportunities to improve the outcome of cancer immunotherapy by coadministration of certain TLR ligands. For example, it has been shown that the suppressive activity of T-reg cells could be reversed by poly-G oligonucleotides, which bind to TLR8 expressed on T-reg cells,23 although this inhibiting effect is only temporary. Apetoh et al24 described a contribution of the immune system to anticancer radiochemotherapy as a clinically relevant immunoadjuvant through a TLR4-dependent activation of tumor antigen–specific T-cell immunity. However, further murine in vivo studies are needed to further delineate the relevance of TLR4 signaling in tumor-mediated immune suppression mediated by T-reg cells and thus evaluate the clinical potential of TLR ligands to reverse T-reg cell function and improve the efficacy of cancer vaccines. Finally, the development and clinical application of combined chemotherapy and immunotherapy may promise enduring success in the fight against cancer.25

Correspondence: Christoph Bergmann, MD, Department of Otorhinolaryngology, University of Duisburg–Essen, Hufelandstr 55, 45122 Essen, Germany (Christoph.bergmann@uk-essen.de).

Submitted for Publication: April 1, 2010; final revision received May 28, 2010; accepted June 29, 2010.

Author Contributions: Dr Bergmann had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Wild, Brandau, Hoffmann, Lang, and Bergmann. Acquisition of data: Wild, Lindemann, and Lotfi. Analysis and interpretation of data: Wild, Brandau, Lotfi, and Bergmann. Drafting of the manuscript: Wild and Bergmann. Critical revision of the manuscript for important intellectual content: Brandau, Lindemann, Lotfi, Hoffmann, Lang, and Bergmann. Statistical analysis: Wild and Bergmann. Obtained funding: Lang and Bergmann. Administrative, technical, and material support: Brandau, Lindemann, Lotfi, and Bergmann. Study supervision: Brandau and Bergmann.

Financial Disclosure: None reported.

Funding/Support: Research described in this article was supported by grant DFG BE/4190 from Deutsche Forschungsgemeinschaft.

Previous Presentation: This study was presented at the annual meeting of the American Head and Neck Society; April 29, 2010; Las Vegas, Nevada.

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Feuerer  MHill  JAMathis  DBenoist  C Foxp3+ regulatory T cells: differentiation, specification, subphenotypes. Nat Immunol 2009;10 (7) 689- 695
PubMed Link to Article
Strauss  LBergmann  CSzczepanski  MGooding  WJohnson  JTWhiteside  TL A unique subset of CD4+CD25highFoxp3+ T cells secreting interleukin-10 and transforming growth factor-β1 mediates suppression in the tumor microenvironment. Clin Cancer Res 2007;13 (15, pt 1) 4345- 4354
PubMed Link to Article
Whiteside  TL The tumor microenvironment and its role in promoting tumor growth. Oncogene 2008;27 (45) 5904- 5912
PubMed Link to Article
Szczepanski  MJCzystowska  MSzajnik  M  et al.  Triggering of Toll-like receptor 4 expressed on human head and neck squamous cell carcinoma promotes tumor development and protects the tumor from immune attack. Cancer Res 2009;69 (7) 3105- 3113
PubMed Link to Article
Netea  MGSutmuller  RHermann  C  et al.  Toll-like receptor 2 suppresses immunity against Candida albicans through induction of IL-10 and regulatory T cells. J Immunol 2004;172 (6) 3712- 3718
PubMed Link to Article
Caramalho  ILopes-Carvalho  TOstler  DZelenay  SHaury  MDemengeot  J Regulatory T cells selectively express Toll-like receptors and are activated by lipopolysaccharide. J Exp Med 2003;197 (4) 403- 411
PubMed Link to Article
Colombo  MPPiconese  S Regulatory T-cell inhibition versus depletion: the right choice in cancer immunotherapy. Nat Rev Cancer 2007;7 (11) 880- 887
PubMed Link to Article
Lang  SWhiteside  TLLebeau  AZeidler  RMack  BWollenberg  B Impairment of T-cell activation in head and neck cancer in situ and in vitro: strategies for an immune restoration. Arch Otolaryngol Head Neck Surg 1999;125 (1) 82- 88
PubMed Link to Article
Wang  R-F Regulatory T cells and Toll-like receptors in cancer therapy. Cancer Res 2006;66 (10) 4987- 4990
PubMed Link to Article
Apetoh  LGhiringhelli  FTesniere  A  et al.  Toll-like receptor 4–dependent contribution of the immune system to anticancer chemotherapy and radiotherapy. Nat Med 2007;13 (9) 1050- 1059
PubMed Link to Article
Zitvogel  LApetoh  LGhiringhelli  FKroemer  G Immunological aspects of cancer chemotherapy. Nat Rev Immunol 2008;8 (1) 59- 73
PubMed Link to Article

Figures

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Figure 1.

Frequency of T-regulatory (T-reg) cells in patients with head and neck squamous cell carcinoma (HNSCC). A, Gating strategy to analyze T-reg and T-effector (T-eff) cells in peripheral blood mononuclear cells of patients with HNSCC or healthy donors (HDs): first, gate was set on lymphocytes according to their forward or sideward scatter properties. Within the CD4+ T-cell population, T-reg cells were identified as CD25+CD127, whereas T-eff cells are represented as CD25CD127+ cells. B, Percentages of CD4+CD25+CD127 T-reg cells of 11 patients with HNSCC and 10 HDs are shown. Data are mean (SD) as determined by flow cytometry.

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Figure 2.

Constitutive expression of Toll-like receptor (TLR) on T-regulatory (T-reg) cells. A, Expression of TLR4, TLR6, TLR9, and TLR10 on T-reg cells isolated from peripheral blood mononuclear cells (PBMCs) of 11 patients with head and neck squamous cell carcinoma (HNSCC) and 10 healthy donors (HDs) as obtained by flow cytometry is shown as mean fluorescent intensity defined as the difference between the staining intensity of the isotype control and the TLR-specific antibody (ΔMFI). Box plots present the mean, median, and interquartile range. B, Histograms illustrate the difference in the expression level of TLR4, TLR6, TLR8, and TLR10 on T-reg cells of patients with HNSCC vs HDs. Flow cytometry histograms shown are representative for all stained PBMCs. Anti–TLR4-FITC-A indicates antibody against TLR4 receptor, conjugated to the FITC fluorochrome; anti–TLR6-FITC-A, antibody against TLR6 receptor, conjugated to the FITC fluorochrome; anti–TLR10-FITC-A, antibody against TLR10 receptor, conjugated to the FITC fluorochrome; and TLR9-PE-A, antibody against TLR9 receptor, conjugated to the PE fluorochrome.

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Figure 3.

Proliferative response of T-effector (T-eff) cells to lipopolysaccharide (LPS) stimulation. T-eff cells were preincubated for 2 hours with LPS at different concentrations as indicated on the x-axis. Cells were washed and activated with αCD2/CD3/CD28 beads. Counts per minute (CPMs) were measured after 72 hours. Thymidine labeled with tritium was added for the last 16 hours. Data shown are mean (SD) CPMs derived from experiments with cells of 4 different donors. Plus signs indicate positive; minus signs, negative.

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Figure 4.

Suppressive capacity of T-regulatory (T-reg) cells on stimulation with heat shock protein 60 (HSP60) or lipopolysaccharide (LPS); P = .04 for all. A, Magnetic associated cell sorter–sorted CD4+CD25+ T-reg cells were preincubated for 2 hours with 0.1-μg/mL LPS or 0.01-μg/mL HSP60, washed extensively, and activated overnight with αCD2/CD3/CD28 beads. On the following day, autologous carboxyfluorescein diacetate succinimidyl ester (CFSE)–labeled CD4+CD25 T-eff (responder) and T-reg (suppressor) cells were seeded at different ratios in U-bottom 96-well plates. Cells were cocultured in the presence of αCD2/CD3/CD28 beads. On day 5, proliferation of responder cells was analyzed using mean fluorescent intensity of CFSE. The data are mean (SD) x-fold suppression compared with untreated T-reg cells in cocultures with responder cells as derived from experiments with cells of 4 different donors. Plus signs indicate positive; minus signs, negative.

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Place holder to copy figure label and caption
Figure 5.

Suppressive activity of T-regulatory (T-reg) cells is mediated through Toll-like receptor 4 (TLR4) but does not confer cytotoxic effects on activated T cells;  = .049 for all. A, Proliferation of T-effector (T-eff) cells was evaluated in carboxyfluorescein diacetate succinimidyl ester (CFSE) suppression cultures. The T-eff cells were labeled with CFSE and activated with αCD2/CD3/CD28 beads in cocultures with T-reg cells for 5 days as described in the “Materials and Methods” section. The T-reg cells were pretreated with lipopolysaccharide (LPS) or with neutralizing antibody against TLR4 as indicated below the graph. The data are mean percentage of proliferation derived from experiments with cells of 4 healthy donors. B, CFSE histograms show 1 representative example of 4 independent experiments. Percentages indicate proliferation of CFSE-positive responder cells (left column) Flow cytometry–based cytotoxicity assay simultaneously measures induction of apoptosis and proliferation of CD4+CD25 T-effector (T-eff) cell proliferation and suppression mediated by T-reg cells. Percentages indicate 7–amino actinomycin D (7-AAD)–positive T-eff cells, which represent cells undergoing apoptosis (right column). Plus signs indicate positive; minus signs, negative.

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Tables

Table Graphic Jump LocationTable. Clinicopathologic Characteristics of Study Patients With Head and Neck Squamous Cell Carcinoma

References

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Argiris  AKaramouzis  MVRaben  DFerris  RL Head and neck cancer. Lancet 2008;371 (9625) 1695- 1709
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Young  MR Protective mechanisms of head and neck squamous cell carcinomas from immune assault. Head Neck 2006;28 (5) 462- 470
PubMed Link to Article
Bluestone  JAAbbas  AK Natural versus adaptive regulatory T cells. Nat Rev Immunol 2003;3 (3) 253- 257
PubMed Link to Article
Vignali  DACollison  LWWorkman  CJ How regulatory T cells work. Nat Rev Immunol 2008;8 (7) 523- 532
PubMed Link to Article
Schaefer  CKim  GGAlbers  AHoermann  KMyers  ENWhiteside  TL Characteristics of CD4+CD25+ regulatory T cells in the peripheral circulation of patients with head and neck cancer. Br J Cancer 2005;92 (5) 913- 920
PubMed Link to Article
Strauss  LBergmann  CGooding  WJohnson  JTWhiteside  TL The frequency and suppressor function of CD4+CD25highFoxp3+ T cells in the circulation of patients with squamous cell carcinoma of the head and neck. Clin Cancer Res 2007;13 (21) 6301- 6311
PubMed Link to Article
Bergmann  CStrauss  LWang  Y  et al.  T regulatory type 1 cells in squamous cell carcinoma of the head and neck: mechanisms of suppression and expansion in advanced disease. Clin Cancer Res 2008;14 (12) 3706- 3715
PubMed Link to Article
Medzhitov  R Toll-like receptors and innate immunity. Nat Rev Immunol 2001;1 (2) 135- 145
PubMed Link to Article
Iwasaki  AMedzhitov  R Toll-like receptor control of the adaptive immune responses. Nat Immunol 2004;5 (10) 987- 995
PubMed Link to Article
Pries  RWulff  SWollenberg  B Toll-like receptor modulation in head and neck cancer. Crit Rev Immunol 2008;28 (3) 201- 213
PubMed Link to Article
Strauss  LBergmann  CWhiteside  TL Human circulating CD4+CD25highFoxp3+ regulatory T cells kill autologous CD8+ but not CD4+ responder cells by Fas-mediated apoptosis. J Immunol 2009;182 (3) 1469- 1480
PubMed Link to Article
Seddiki  NSantner-Nanan  BMartinson  J  et al.  Expression of interleukin (IL)-2 and IL-7 receptors discriminates between human regulatory and activated T cells. J Exp Med 2006;203 (7) 1693- 1700
PubMed Link to Article
Feuerer  MHill  JAMathis  DBenoist  C Foxp3+ regulatory T cells: differentiation, specification, subphenotypes. Nat Immunol 2009;10 (7) 689- 695
PubMed Link to Article
Strauss  LBergmann  CSzczepanski  MGooding  WJohnson  JTWhiteside  TL A unique subset of CD4+CD25highFoxp3+ T cells secreting interleukin-10 and transforming growth factor-β1 mediates suppression in the tumor microenvironment. Clin Cancer Res 2007;13 (15, pt 1) 4345- 4354
PubMed Link to Article
Whiteside  TL The tumor microenvironment and its role in promoting tumor growth. Oncogene 2008;27 (45) 5904- 5912
PubMed Link to Article
Szczepanski  MJCzystowska  MSzajnik  M  et al.  Triggering of Toll-like receptor 4 expressed on human head and neck squamous cell carcinoma promotes tumor development and protects the tumor from immune attack. Cancer Res 2009;69 (7) 3105- 3113
PubMed Link to Article
Netea  MGSutmuller  RHermann  C  et al.  Toll-like receptor 2 suppresses immunity against Candida albicans through induction of IL-10 and regulatory T cells. J Immunol 2004;172 (6) 3712- 3718
PubMed Link to Article
Caramalho  ILopes-Carvalho  TOstler  DZelenay  SHaury  MDemengeot  J Regulatory T cells selectively express Toll-like receptors and are activated by lipopolysaccharide. J Exp Med 2003;197 (4) 403- 411
PubMed Link to Article
Colombo  MPPiconese  S Regulatory T-cell inhibition versus depletion: the right choice in cancer immunotherapy. Nat Rev Cancer 2007;7 (11) 880- 887
PubMed Link to Article
Lang  SWhiteside  TLLebeau  AZeidler  RMack  BWollenberg  B Impairment of T-cell activation in head and neck cancer in situ and in vitro: strategies for an immune restoration. Arch Otolaryngol Head Neck Surg 1999;125 (1) 82- 88
PubMed Link to Article
Wang  R-F Regulatory T cells and Toll-like receptors in cancer therapy. Cancer Res 2006;66 (10) 4987- 4990
PubMed Link to Article
Apetoh  LGhiringhelli  FTesniere  A  et al.  Toll-like receptor 4–dependent contribution of the immune system to anticancer chemotherapy and radiotherapy. Nat Med 2007;13 (9) 1050- 1059
PubMed Link to Article
Zitvogel  LApetoh  LGhiringhelli  FKroemer  G Immunological aspects of cancer chemotherapy. Nat Rev Immunol 2008;8 (1) 59- 73
PubMed Link to Article

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