Interleukin 17 and peripheral IL-17-expressing T cells are negatively correlated with the overall survival of head and neck cancer patients

Abstract

The presence and clinical significance of interleukin (IL)-17 and IL-17-expressing cells have recently been studied in several types of cancer, but their correlation to tumor development remains controversial. Additionally, the contribution of peripheral IL-17-expressing cells to head and neck cancer (HNC) progression is still poorly understood. We collected peripheral blood from healthy donors and HNC patients to isolate PBMCs. The percentages of IL-17-expressing cells and the production of inflammatory cytokines in PBMCs were measured to determine their association with clinical outcomes and overall survival in HNC. We evaluated the effect and potential mechanism of IL-17 on human oral squamous carcinomas in vitro using exogenous IL-17 stimulation. In comparison to healthy donors, the PBMCs of HNC patients have a significant accumulation of IL-17-expressing T cells and their frequencies were positively correlated with the disease stage. A significantly higher production of PBMC IL-17, TGF-β and IL-21 and plasma VEGF-A were found in HNC patients. Importantly, the 5-years overall survival of HNC patients with a higher percentage of IL-17-expressing cells is significantly decreased. Furthermore, the addition of IL-17 appeared to promote human oral squamous carcinoma cell proliferation via the production of IL-6 and VEGF-A. Our findings suggest that IL-17 has the potential to mediate pro-tumor immunity in the HNC tumor microenvironment. Enhanced IL-17-expressing cells, including Th17 and Tc17 cells, in the peripheral blood could be a significant predictor of a poor prognosis for HNC patients.

Keywords: PBMCs; Th17 cells; head and neck cancer; interleukin-17; prognosis.

Figure 1. The frequency of IL-17-expressing T cells increases in the peripheral blood of patients with HNC

Isolated PBMCs from healthy donors and patients with HNC and the percentage of IL-17-expressing cells, Th17 and Tc17, was evaluated by flow cytometric analysis. (A) Representative plots for identification of IL-17 producing cells in PBMC, CD4+ or CD8+ populations. (B–C) Analysis of the frequency of IL-17 producing cells from the PBMCs of healthy donors in comparison to HNC patients with tumors at different stages. Each dot represents an individual sample.

Figure 2. The expression of peripheral blood IL-17-producing T cells is negatively associated with 5-year survival for HNC patients

We correlated expression of peripheral blood IL-17-producing T cells in HNC patients with their survival rates after surgery. 72 HNC patients were divided into 2 groups based on their median levels (%) of IL-17 producing cells. The 5-year overall survival rates for HNC patients with a high and low level of (A) Th17, (B) Tc17, and (C) IL-17⁺ cells in their PBMCs and (D) IL-17 from PBMC culture supernatant. The survival rates were determined using the Kaplan-Meier method (log-rank test).

Figure 3. IL-17 induces the proliferation of oral squamous carcinoma cells (OSCC) proliferation through the production of IL-6

(A) The increased expression of IL-17RA was analyzed by immunohistochemistry in a tissue array of human oral squamous cell cancer and adjacent normal tongue tissues (200x magnification). (B) IL-17RA expression in OSCC cell lines (SAS, OECM-1 and OC3) was analyzed by qRT-PCR assay (upper panel) and western blot analysis (lower panel). (C) OSCC cell lines were then treated with 100 ng/mL rhIL-17 for 48 h and the proliferation rates analyzed by cell counting using trypan blue exclusion. (D) The expression levels of Pcna, Ki67, Il-6, Vegf-a and Mmp2 from IL-17-stimulated OSCC cell lines were measured by qRT-PCR and graphed as relative fold changes from untreated cells. (E) The OSCC culture supernatants were analyzed for protein levels of IL-6 after treatment with rhIL-17 for 48 h using ELISA. (F) OSCC cells were treated with or without different concentrations of rhIL-6 (0.125-2 ng/mL) for 48 h, and cell proliferation was determined by MTT assay. All results represent the mean ± SEM of at last three independent experiments.

Figure 4. The high expression of IL-17-producing T cells was positively correlated with VEGF-A production in HNC and to facilitate OSCC cells proliferation

(A) OSCC cells were cultured in the presence of 100 ng/mL rhIL-17 for 48 h and the VEGF-A content from culture supernatants were analyzed for by ELISA. (B) OSCC cells were treated with or without different concentrations of recombinant VEGF-A (0.25-2 ng/mL) for 48 h, and cell proliferation was determined by MTT assay. (C) Correlations between the level of IL-17-producing T cells (Th17 and Tc17) and plasma VEGF-A in HNC patients (n = 84) were analyzed by Pearson correlation test. (D) The production of VEGF-A in plasma from HNC patients (early and late stage) and healthy donors were analyzed by ELISA. Data are representatives of at least three experiments and shown as the mean ± SEM.