1. Date of download: 5/31/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Toll-like Receptors in Regulatory T Cells of Patients With Head and Neck Cancer Arch Otolaryngol Head Neck Surg. 2010;136(12):1253-1259. doi:10.1001/archoto.2010.195 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 CD25 − CD127 + 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. Figure Legend:

2. Date of download: 5/31/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Toll-like Receptors in Regulatory T Cells of Patients With Head and Neck Cancer Arch Otolaryngol Head Neck Surg. 2010;136(12):1253-1259. doi:10.1001/archoto.2010.195 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. Figure Legend:

3. Date of download: 5/31/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Toll-like Receptors in Regulatory T Cells of Patients With Head and Neck Cancer Arch Otolaryngol Head Neck Surg. 2010;136(12):1253-1259. doi:10.1001/archoto.2010.195 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. Figure Legend:

4. Date of download: 5/31/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Toll-like Receptors in Regulatory T Cells of Patients With Head and Neck Cancer Arch Otolaryngol Head Neck Surg. 2010;136(12):1253-1259. doi:10.1001/archoto.2010.195 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. Figure Legend:

5. Date of download: 5/31/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Toll-like Receptors in Regulatory T Cells of Patients With Head and Neck Cancer Arch Otolaryngol Head Neck Surg. 2010;136(12):1253-1259. doi:10.1001/archoto.2010.195 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; P =.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. Figure Legend: