Materials and methods Patients and healthy donors From September 2012 to February 2014, 112 HNSCC patients were enrolled in the present study [19 oral cavity squamous cell carcinoma (OCSCC), 20 hypopharyngeal squamous cell carcinoma (HPSCC), 18 nasopharyngeal squamous cell carcinoma (NPSCC), 19 oropharyngeal squamous cell carcinoma (OPSCC),
and 36 laryngeal squamous cell carcinoma (LSCC)]. Patients were diagnosed at the Department of Otorhinolaryngology, the First Affiliated Hospital of Sun Yat-sen University without any previous oncological treatment. Healthy SC79 age-matched donors (29 males and 2 female with a mean age of 45 years; range: 38–81) were enrolled as controls. The main clinical and pathologic characteristics of the patients are presented in Table 1. Clinical staging and the anatomic subsites
of the tumors were assessed according to the 6th edition of the Union Internationale Contre Cancer (UICC 2008) tumor-node-metastasis classification of malignant tumors. Table 1 Clinicopathological features of 112 HNSCC patients who donated peripheral blood for this study Characteristics Number Age (years) mean (range) 47 (37–83) Gender Male 108 Female 4 Total 112 Tumor site Oral cavity 19 Hypopharynx 20 Nasopharynx 18 Oropharynx 19 Larynx 36 Tumor stage T1–2 46 T3–4 66 Nodal status N0 70 N+ 42 M stage M0 112 M1 0 HNSCC, Head and neck squamous cell carcinoma. Ethics statements The study protocol PF-6463922 molecular weight (No. 2012–349) was approved by the ethic Committee of The First Affiliated Hospital of Sun Yat-sen University,
and was used for research purposes only. Patient and healthy donor (HD) informed consent was obtained before enrollment. Collection of peripheral blood Peripheral blood lymphocytes (PBLs) were isolated from peripheral venous blood as previously described . Isolated cells were immediately DNA Damage inhibitor re-suspended in 100 μl flow cytometry staining buffer (eBioscience, San Diego, CA, USA) for surface and intracellular staining. Antibodies and reagents Freshly obtained human PBLs were stained with the following anti-human monoclonal clonidine antibodies: anti-CD3-eFluor 605NC (0.25 μg/100 μl), anti-CD4-FITC (1.0 μg/100 μl), anti-CD25-APC (0.125 μg/100 μl), and anti-CD45RA-eFluor 450 (0.5 μg/100 μl) for surface staining. Anti-Foxp3-PE (0.25 μg/100 μl), anti-tumor necrosis factor-alpha (TNF-α)-Alexa Fluor 700 (0.25 μg/100 μl), anti-interleukin-2 (IL-2)-PE-Cy7 (0.125 μg/100 μl), anti-interferon-gamma (IFN-γ)-APC-eFluor780 (0.25 μg/100 μl), and anti-hinterleukin-17 (IL-17)-PerCP-Cy5.5 (0.125 μg/100 μl) for intracellular staining. Soluble anti-CD3 (OKT3, 0.5 μg/ml) and anti-CD28 (CD28.2, 2 μg/ml) mAb were used for in vitro activation of T cells. All antibodies and isotype controls were purchased from eBioscience (San Diego, CA, USA).