The Accumulation and Prognosis Value of Tumor Infiltrating IL-17 Producing Cells in Esophageal Squamous Cell Carcinoma

Background The role of IL-17 producing cells in tumors is controversial. In the present study, we investigated the prognostic value of measuring tumor-infiltrating IL-17 producing cell levels in human esophageal squamous cell carcinoma (ESCC). Methodology/Principal Findings Immunohistochemical staining was performed to investigate the levels of IL-17+ tumor infiltrating lymphocytes (TILs), as well as CD8+ cytotoxic T lymphocytes (CTLs) and CD57+ natural killer (NK) cells from 181 ESCC patients. The prognostic value of measuring the densities of IL-17+TILs and the correlation with CTLs and NK was evaluated. IL-17 producing cells were detected in esophageal squamous cell carcinoma tissues. The IL-17 producing cells were major CD4 positive, but Foxp3 negative. The median level of IL-17+TILs was 3.90 cells/high power microscopic field (HPF). The density of IL-17 producing cells correlated negatively with T stage (P = 0.042). The higher densities of tumor infiltrating IL-17+ lymphocytes were associated with better overall survival (P = 0.031). Furthermore, we found that there were positive correlations between levels of IL-17 producing cells and the densities of CD8+cells, as well as CD57+cells (r = 0.198, P = 0.008 for CD8+ cells and r = 0.261, P<0.001 for CD57+ cells, respectively). The prognosis analysis also showed that the higher levels of CD8+ CTLs and CD57+ NK cells correlated with better overall survival of ESCC patients. Conclusions Our study suggests that tumor infiltrating IL-17 producing cells in ESCC patients may have protective roles in the tumor microenvironment and may be treated as a prognostic marker for ESCC patients.


Introduction
Substantial evidence indicates that the abundance of tumorinfiltrating lymphocytes in the microenvironment of certain tumor types is associated with the prognosis of cancer patients. Moreover, each subset of tumor-infiltrating lymphocytes has a unique role in the antitumor response [1][2][3][4]. The presence of tumor-infiltrating cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells correlates with improved survival and confers antitumor activity [5]. However, other tumor-infiltrating lymphocyte subsets exhibit bipolar roles: promoting tumor growth or inhibiting tumor progression [6]. These subsets include the newly identified tumor-infiltrating IL-17 producing cells. Interleukin-17 (IL-17), originally termed CTLA-8, plays an important role in inflammation and autoimmune diseases in both mice and humans [7][8][9][10][11][12][13]. Early research focused on the roles and mechanisms of IL-17 producing cells in inflammation and autoimmune diseases. Because chronic inflammation were correlated significantly to tumor invasion, migration and metastasis [14,15], scientists have begun to pay more attention to the significance of IL-17 in tumor models. There is accumulating evidence that IL-17 producing cells are present in various cancers, including ovarian cancer, breast cancer, non-small cell lung cancer, hepatocellular carcinoma and gastric cancer [16][17][18][19]. Substantial evidence indicated that IL-17 was produced mainly by CD4+ T lymphocytes, and these cells were defined as T helper 17 (Th 17) cells [14,15,20]. However, in recent studies, it was found that other T cell subsets can also produce IL-17, such as NKT, gamma-delta T cells and Tregs, including mouse models and human beings [14,[20][21][22][23][24][25][26]. Although IL-17 producing cells have been detected in various tumors, their effect on tumor cell survival and exact physiological role in tumor immunity remain controversial. IL-17 producing cells could enhance tumor growth by promoting angiogenesis [14,18]. Conversely, IL-17 producing cells might promote tumor regression by enhancing antitumor immunity [15,[27][28][29][30].
Esophageal squamous cell carcinoma (ESCC) is the major histological type of esophageal cancer in the "Esophageal Cancer Belt," which stretches westward from China through central Asia to northern Iran [31,32]. ESCC is the eighth most common cancer worldwide [33], and ranks the sixth cancer mortality worldwide [34]. It was reported that the host immune response prompted by ESCC may influence patient prognosis; both adaptive and innate immunity play important roles in ESCC progression and regression [35][36][37][38]. Yasushi et al showed that the number of CD8+ T cells correlated with favorable outcomes in ESCC patients [39]. Hsia et al found that ESCC patient prognosis correlated positively with intratumoral NK cell infiltration [36]. Xue et al found that FOXP3 expression was associated with lymph node metastasis and pathological TNM staging, suggesting that regulatory T cells (Tregs) might promote tumor progression [38]. However, up until now, the presence and clinical significance of IL-17 producing cells have not been previously studied in ESCC. Thus, in this study, we evaluated the accumulation and clinicopathological significance of tumor-infiltrating IL-17 producing cells in tumor tissues from ESCC patients. The prognosis value of IL-17 producing cells was also evaluated. Furthermore, we detected CD8+ CTLs and CD57+ NK cells in the same tumor tissues and relationships between the number of IL-17 producing cells and the density of CD8+ CTLs or CD57+ NK cells were further evaluated.

Results
Immunohistochemical staining of IL-17 producing cells and their associations with clinicopathological characteristics The representative photomicrographs of tissue sections immunostained for IL-17 are shown in Figure 1. IL-17 producing cells were detected in esophageal squamous cell carcinoma tissue. In order to clarify which set of T cells could produce IL-17, double immunohistochemical staining was performed in the same tissue section. Immunofiuorescence results showed that IL-17 producing cells were major CD4 positive, but FOXP3 negative in ESCC tissues ( Figure 1). The median value of IL-17 producing cells was 3.90 cells/HPF (range: 0.00-21.40 cells/HPF).
The associations between the levels of IL-17 producing cells and clinicopathological factors of the ESCC patients are summarized in Table 1. According to previous studies, patients were divided into two groups based on the median of IL-17 producing cells (high level group vs. low level group) [4,18]. There was a significant inverse correlation between the densities

Correlation between the densities of IL-17 producing cells and patients' survival
The median survival time of the 181 ESCC patients was 44 months (range 1-87 months). The five-year survival rate was 49.9%. The overall survival curves of the patients in this study are depicted in Figure 2. The statistical analysis demonstrated a positive correlation between overall survival and the density of IL-17+ TILs (Figure 2, long-rank test: P = 0.031).

Relationship between the levels of IL-17+ TILs and CD8+ CTLs cells as well as CD57+ NK cells in tumor microenvironment
The levels of CD8+ CTLs and CD57+ NK cells in tumor tissue in ESCC patients were evaluated. In addition, the Pearson correlation coefficient was calculated, and linear regression analysis was applied to assess the relationships between the IL-17 producing cells and CD8+ CTLs or CD57+ NK cells in the tumor microenvironment. Infiltrating CD8+ CTLs and CD57+ NK cells were detected in tumor tissues by immunohistochemical staining (Figure 3), and statistical analysis showed that the levels of IL-17 producing cells positively correlated with levels of CD8+ CTLs in tumor tissues (r = 0.198, P = 0.008, Figure 4A). A significant association was also identified between the densities of IL-17+ TILs and CD57+ NK cells (r = 0.261, P,0.001, Figure 4B). From prognosis analysis, we found that higher levels of infiltrating CD8+ CTLs or CD57+NK cells correlated with a better overall survival of ESCC patients (P,0.001 and P = 0.002, respectively, determined by long-rank test, Figure 5).

Discussion
Accumulating evidence suggests that IL-17 producing cells play a significant role in tumor immunity. However, IL-17 may play pro-tumor or anti-tumor effects in different tumor contexts [14,18,27,28,40]. Zhang et al found that IL-17 producing cells could promote tumor growth by stimulating angiogenesis in hepatocellular carcinoma patients [18]. Conversely, Kryczek et al demonstrated that IL-17 promoted antitumor activity in ovarian cancer patients [28]. The explanation for this discrepancy remains unknown.
We first detected tumor-infiltrating-IL-17 producing cells in human esophageal squamous cell carcinoma (ESCC) and observed that the levels of IL-17 producing cells correlated inversely with T    [28]. Thus, in ESCC, our results suggest that IL-17 producing cells may mediate antitumor immunity.
We also detected CD8+ CTLs and CD57+ NK cells in tumor tissues from ESCC patients using serial tissue sections. Our study found that infiltrating CD8+ CTLs cells and CD57+ NK cells were present in ESCC tumors, and the abundance of CD8+ T or CD57+ cells correlated positively with the number of IL-17 producing cells. Martin-Orozco et al reported that Th17 cells can induce a strong antitumor CD8 response by eliciting the priming and recruitment of CD8+ T cells in a mouse model of lung melanoma [30]. Benchetrit et al found that IL-17 could inhibit tumor growth by inducing tumor-specific cytotoxic T lymphocyte (CTL) activity in hematopoietic tumors in immunocompetent mice [27]. In addition, Kryczek et al reported that both natural killer cell-mediated innate immunity and tumor-specific T-cell immunity were weakened in IL-17 deficient mice bearing MC38 tumors [29]; they also demonstrated that the abundance of IL-17 producing cells correlated positively with CD8+ T and NK cells in the same tumor microenvironment [28]. Our results are consistent with these studies, and indicate that IL-17 producing cells in ESCC might exert antitumor effects by enhancing cytotoxic T lymphocytes and NK cell responses.
In conclusion, our data show that IL-17 producing TILs were detected in ESCC, and the density of IL-17+ TILs was associated with better overall survival. Furthermore, the number of IL-17 producing cells correlated positively with the numbers of CD8+ CTLs and CD57 + NK cells. Thus, tumor infiltrating IL-17 producing cells may constitute a novel prognosis marker for ESCC  and may play an antitumor role by activating innate and adaptive immunity.

Patients and tissue samples
Paraffin-embedded samples were obtained from 181 ESCC patients who underwent surgery at the Sun Yat-sen University Cancer Center between 2002 and 2003. There were 141 male and 40 female patients with a median age of 56 years (range, 33-79 years). Patients with autoimmune diseases and other esophageal cancers (e.g., adenocarcinoma) were excluded. None of the patients had received anticancer treatment prior to surgery. The follow-up data from the ESCC patients in this study are available and complete. There were 117 cases of stage I-II and 64 cases of stage III-IV cancer according to the American Joint Committee on Cancer (AJCC, 2002) TNM staging system. Each lesion was graded histologically according to the WHO classification criteria. Overall survival (OS) was defined as the interval between the date of surgery and date of death or the last known follow-up. The study was approved by the Ethics Committee of Sun Yat-sen University Cancer Center, and informed consent was obtained from each patient.
Data were obtained by manually counting positively stained cells in ten separate fields under 4006 high power magnification. The density of stained cells was determined by computing the mean number of positively stained cells per high power microscopic field (HPF).

Statistical analysis
Quantitative values were expressed as means 6 SD or median (range). Patients were divided into two groups based on the median of various immunohistochemical variables in our data (high level group vs. low level group). The Chi-square test or Fisher exact test was used to assess the relationships between the levels of IL-17 producing cells and clinicopathological features. The overall survival curves were calculated using the Kaplan-Meier method The survival rate for patients in the high CD8+ CTLs density group was significantly better than that for patients in the low density group (log rank test, P,0.001). (B) Kaplan-Meier survival curves for high CD57+ NK cell density group versus low CD57+ NK cell density group showed a highly significant separation (log-rank test: P = 0.002). doi:10.1371/journal.pone.0018219.g005 and analyzed using the long-rank test. Prognostic factors were examined by univariate and multivariate analyses using the Cox proportional hazards model. The correlation between the density of IL-17 producing cells and CD8+ CTLs or CD57+ NK cells were determined by Pearson correlation coefficient and linear regression analyses. A two-tailed P-value,0.05 was considered statistically significant. All statistical analyses were performed with SPSS software (version 16.0; SPSS Inc., Chicago, IL, USA).

Author Contributions
Conceived and designed the experiments: J-cX KP. Performed the experiments: LL KP. Analyzed the data: LL J-jZ WW J-gC J-pY. Contributed reagents/materials/analysis tools: X-dL K-lS J-pY Y-bC WW. Wrote the paper: LL KP J-cX.