The Ethics Committee of Second Xiangya Hospital ratified this study. Written informed consents were obtained from all participants prior to enrollment. All experimental methods abided by the Helsinki Declaration.
Patients and Specimens
All OSCC samples were collected from Department of Oral and Maxillofacial Surgery, Second Xiangya Hospital of Central South University. More specifically, lymph nodes were obtaied from the OSCC patients who underwent surgery treatment between June 2018 and July 2019. Half of each lymph node was stored for experiments, and other half was sent for pathological diagnosis. The clinical parameters were obtained from medical records.
RNA extraction and real-time PCR (RT-PCR) analysis
Total RNA of CD4+ T cells were isolated by TRIzol reagent (Takara, Japan) and cDNA was synthesized with a PrimeScript RT Reagent Kit (Takara, Japan). Real-time PCR was applied using SYBR Premix Ex Taq Reagent Kit (Takara, Japan) by the StepOne Real-Time PCR System (Life Technologies, USA) according to manufacturer's instructions. In tissue lysates, the mRNA levels were normalized against β-actin levels. The primer sequences used in the present study are listed in Supplementary Table 1.
Briefly, Paraffin-embedded sections were deparaffinized, rehydrated and submerged into EDTA buffer for heat-induced antigen retrieval. Then, the sections immersed into 0.3% hydrogen peroxide, blocked with 10% goat serum, incubated with specific primary antibodies at 4°C overnight, and incubated the Alexa Fluor 488-cojugated secondary antibody (Invitrogen, USA) or Alexa Fluor 549-cojugated secondary antibody (Invitrogen, USA) in the dark at room temperature. Sections stained with DAPI (Sangon Biotech, China) to detect nuclei. Sections were imaged using a TCS SP2 laser-scanning confocal microscope (Leica Microsystems, Germany) and Gen5 software (Bio Tek, USA).
The cell surface markers were analyzed by flow cytometry (FCM). The living cells were stained with antibodies in PBS containing 0.1% (weight/volume) BSA and 0.1% NaN3 in 50μL FACs buffer for 30min on ice. The following antibodies-fluorochrome combinations were used: anti-CD4 BB515 (RPA-T4), anti-CD8a BB700 (RPA-T8), anti-CD19 Percp (HIB19), anti-CD20 FITC (2H7), anti-CD11c PE (3.9), anti-MHCII Percp (G46-6), anti-CD68 FITC (Y1/82A), anti-CD86 Percp (FUN-1), anti-CD274 FITC (MIH1), anti-CD279 APC ( EH12.2H7), anti-CD152 APC (BNI3). The antibodies were obtained from BioLegend or BD Pharmingen.
T cell isolation
CD4+ T cells were sorted from a single-cell suspension drawn from lymph node with the CD4+ T cell Isolation Kit (Biolegend), and purity levels were greater than 95%, as determined by using the BD FACSCalibur. CD4+ T cells were used for Real-time PCR analysis.
Briefly, the OSCC tissue were fixed, paraffin-embedded, sectioned, dehydrated and, then the sections were incubated with anti-KRT14 (1:100, Abcam) at 4 °C overnight, followed by DAKO ChemMate envision kit/HRP (Dako-Cytomation, USA) according the manufacturer’s instructions. Pictures were taken with light microscopy (Olympus, Tokyo, Japan) and Gen5 software (Bio Tek, USA).
Kruskal-Wallis tests, Mann-Whitney tests or nonparametric paired test (Wilcoxon matched paired test) were used to analyze the non-parametric distribution of samples. All statistical analyses were calculated using SPSS 17.0 (SPSS, Chicago, IL, USA). All values were two sided, and p < 0.05 was considered to be significant.