Abstract

Introduction

Substantial evidence indicates that the abundance of tumor-infiltrating 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]–[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]–[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]–[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]–[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]–[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]–[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).

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Figure 1

Representative immunohistochemical staining photomicrographs of IL-17 producing cells in tumor tissues of ESCC.

Positive staining of IL-17 was detected in cellular cytoplasm of tumor tissues. (A), (B), (E) and (F): Low density of IL-17+ TILs. (C), (D), (G) and (H): High density of IL-17+ TILs. (J) and (K): Double stainings of CD4 (green, on the menbrane) and IL-17 (red, in the cytoplasm), IL-17 (green, in the cytoplasm) and FOXP3 (red, nuclei) in paraffin-embedded specimens were analyzed by immunofiuorescence. Original magnification: A–D×200; E–K×400.

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 of IL-17+ TILs and T (depth of primary tumor invasion, P=0.042).

Table 1

Relationship between IL-17 producing cell density and clinical-pathologic factors.

Variables	Number of Patients	IL-17 producing cell density		P value
		Low level group, n=90	High level group, n=91
Age(years)				0.506
<60	105	50	55
≥60	76	40	36
Gender				0.080
Male	141	75	66
Female	40	15	25
Tumor length (cm)				0.110
<5	75	32	43
≥5	106	58	48
Differentiation				0.199
G1	45	26	19
G2	85	39	46
G3	51	25	26
Location				0.952
Upper third	13	6	7
Middle third	113	56	57
Lower third	55	28	27
T				0.042*
T1+T2	57	22	35
T3+T4	124	68	56
N				0.335
No	101	47	54
Yes	80	43	37
M				0.444
M0	175	86	89
M1	6	4	2
TNM staging				0.177
Stage I-II	117	53	64
Stage III-IV	64	37	27

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G1, well differentiated; G2, moderately differentiated; G3, poorly differentiated; T, depth of primary tumor invasion; N, regional lymph nodes; M, distant metastasis; TNM, tumor-lymph node-metastasis classification.

* Statistically significant (P<0.05).

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).

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Figure 2

Kaplan–Meier survival curves of ESCC patients (n=181) after surgical resection.

Increased IL-17-producing cells correlate with improved patient survival. Patients in high IL-17-producing cell density group exhibited significantly better survival than the low IL-17-producing cell density group (log-rank test: P=0.031).

The univariate analysis demonstrated that IL-17 producing cell density (P=0.006), differentiation (P=0.006), T (depth of tumor invasion, P=0.002), N (lymph node metastases, P<0.001) and TNM staging (P<0.001) were significantly associated with overall survival (Table 2). The subsequent multivariate analysis, however, indicated that only differentiation was an independent predictor for overall survival in ESCC (P=0.030, Table 2).

Table 2

Univariate and multivariate analyses of variables associated with overall Survival.

Variables	Univariate analysis			Multivariate analysis
	HR	95% CI	P value	HR	95% CI	P value
IL-17+TIL (high vs. low)	0.633	0.416–0.964	0.033*	0.662	0.431–1.015	0.058
Age, (≥60 vs. <60)	1.141	0.752–1.731	0.535
Gender (female vs. male)	0.813	0.490–1.351	0.425
Location(lower/middle/upper)	1.093	0.754–1.585	0.638
Length, (≥5 vs. <5)	1.226	0.801–1.876	0.348
Differentiation (G3/G2/G1)	1.510	1.123–2.032	0.006*	1.384	1.033–1.856	0.030*
T (T3+T4 vs. T1+T2)	2.233	1.330–3.750	0.002	1.605	0.875–2.942	0.126
N (Yes vs. No)	3.235	2.103–4.979	<0.001*	2.176	1.001–4.728	0.050
M (M1 vs. M0)	2.328	0.944–5.741	0.067
TNM (III+IV vs. I+II)	3.477	2.282–5.298	<0.001*	1.433	0.632–3.251	0.389

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* Statistically significant (P<0.05).

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).

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Figure 3

Representative photomicrographs showing immunohistochemical staining of IL-17, CD8 and CD57 in the same ESCC tissues.

(A) Low density of IL-17 producing cells. (B) Low density of CD8+ CTLs cells. (C) Low density of CD57+ NK cells. (D) High density of IL-17 producing cells. (E) High density of CD8+ CTLs cells. (F) High density of CD57+ NK cells (original magnification ×400).

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Figure 4

IL-17 producing cells correlated positively with the densities of CD8+ CTLs and CD57+ NK cells.

The correlation between the densities of IL-17 producing cells and CD8+T lymphocytes (A), CD57+NK cells (B). The samples were divided into two groups based on the median value of IL-17 producing cells.

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Figure 5

Kaplan–Meier survival curves of ESCC patients (n=181) after surgical resection.

Increased tumor-infiltrating CD8+ CTLs and CD57+ NK cells predict improved patient survival. (A) 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).

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 (depth of primary tumor invasion, P=0.042), indicating that enriched IL-17 producing cells in the tumor microenvironment may inhibit tumor invasion. Kaplan-Meier analysis revealed that increased levels of IL-17 producing cells were linked to better overall survival in ESCC patients (Figure 2, P=0.031), indicating that IL-17 producing cell levels could potentially serve as a prognostic marker for ESCC. Our results are consistent with those of Kryczek et al, who demonstrated that the presence of IL-17+TILs correlated with favorable outcome in and enhanced survival of ovarian cancer patients [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.

Materials and Methods

Footnotes

References