Tim-3 Expression by Peripheral Natural Killer Cells and Natural Killer T Cells Increases in Patients with Lung Cancer-Reduction after Surgical Resection

Lung cancer is one of the most common and deadly malignant tumors worldwide, with high mortality rate, and still remaining a growing trend every year (Liu et al., 2013; Lu et al., 2013). Studies have shown that the development and progression of lung cancer are closely related with immunological dysfunction, especially cell-mediated immunity (Wang et al., 2013). Natural killer (NK) cells and natural killer T (NKT) cells are critical components of cellular immunity that are the first line of defense against chronic viral infection and tumor evasion, and involved in the amplification of the immune response to pathogens and cancer cells. In recent years, studies have demonstrated that T cell immunoglobulin and mucin domain 3 (Tim-3) is closely associated with dysregulation of innate immune cells, including macrophages/monocytes (Yang et al., 2013), dendritic cells (Chiba et al., 2012), and NK cells (Gleason et al., 2012). Abnormal expression of Tim-3 on these innate immune cells was found to be involved in the progression of many clinical diseases (Wu et al., 2012; Anderson et al., 2012; da Silva et al., 2014; Meggyes et al., 2014). However, it was not reported the relationship between Tim-3 expression on NK cells and NKT cells Abstract


Introduction
Lung cancer is one of the most common and deadly malignant tumors worldwide, with high mortality rate, and still remaining a growing trend every year (Liu et al., 2013;Lu et al., 2013).Studies have shown that the development and progression of lung cancer are closely related with immunological dysfunction, especially cell-mediated immunity (Wang et al., 2013).Natural killer (NK) cells and natural killer T (NKT) cells are critical components of cellular immunity that are the first line of defense against chronic viral infection and tumor evasion, and involved in the amplification of the immune response to pathogens and cancer cells.In recent years, studies have demonstrated that T cell immunoglobulin and mucin domain 3 (Tim-3) is closely associated with dysregulation of innate immune

Tim-3 Expression by Peripheral Natural Killer Cells and Natural Killer T Cells Increases in Patients with Lung Cancer -Reduction after Surgical Resection
Li-Yun Xu1 , Dong-Dong Chen 1 , Jian-Ying He 1 , Chang-Chang Lu2 , Xiao-Guang Liu 1 , Han-Bo Le3 , Chao-Ye Wang 3 , Yong-Kui Zhang 2 * and lung cancer.This study is designed to examine the expression of Tim-3 on peripheral NK cells and NKT cells in lung cancer patients using flow cytometry, and to analyze the relationship between disease progression and Tim-3 expression.Furthermore, Tim-3 expression was also analyzed after surgery in a consecutive subset of patients to determine the effect of tumor debulking.

Human subjects and blood sample preparation
The study group included 79 lung cancer patients and 53 healthy volunteer donors from Zhoushan Hospital.The diagnosis of lung cancer was confirmed by histopathological examination.The healthy population was recruited from people who came for general health examination, and they were confirmed to be without any malignant disease and other diseases.Blood samples were obtained from patients with lung tumors before surgery or healthy volunteer donors and processed within 6 hours of collection.Postoperative sample collection was added late in the study, and these samples were collected consecutively.The postoperative sample stage distribution is 8 patients with I/II, and 13 patients with stage III/IV.

Staining and flow cytometric analysis
Every 50 µl peripheral blood sample was incubated for 30 min in 4℃ in a dark room with 5 µl monoclonal antibodies or isotype matched control.Allophycocyanin (APC)-conjugated anti-human Tim-3 (Catalog Number. FAB2365A, Clone Number. 344823 eBM2a, eBioscience, USA) were used for flow cytometric analysis according to the manufacture's instructions.50 µl peripheral blood sample was submitted to 2 ml Red Blood Cell lysis using 1:10 FACS lysis solution (BD, Biosciences, San Jose, CA) and incubated for 10 min at room temperature.Centrifugation was set as 1200 rpm/min for 5 min and washed for three times with phosphate buffer saline (PBS).Then 500 µl PBS was added.At least 30, 000 cells were analyzed using a BD FACSCalibur TM Flow Cytometer (BD Biosciences) and the data were analyzed using the FlowJo software (TreeStar Inc, Ashland, OR, USA).

Statistical analysis
All data were analyzed using the GraphPad Prism 5 (GraphPad Software, Inc.).The student t-test and a paired Wilcoxon rank-sum test were used for comparison.Value of p<0.05 is considered as a significant difference.

Characteristics of the study population
The distribution of selected characteristics of the cases and healthy controls are presented in Table 1.79 lung cancer cases and 53 healthy controls did not reveal any significant difference in age (p>0.05),gender (p>0.05) and smoking history (p>0.05).Among 79 lung cancer patients, 40 (50.6%)cases were size of 0~3 cm and 39 (49.4%) were size of >3 cm.The histological type of 52 (65.8%) patients was adenocarcinoma, whereas squamous cell carcinoma accounted for 27 (34.2%).The Union for International Cancer Control/American Joint Committee on Cancer TNM Classification (7 th ed) was also shown, in which 37 (46.8%) were in stage I ~ II and 42 (53.2%)were in stage III~IV.

Tim-3 expressions on peripheral CD3-CD56+ NK cells and CD3+CD56+ NKT cells in lung cancer
Although Tim-3 was first cloned in differentiated Th1 cells, studies have indicated that Tim-3 expression on various immune cells, such as CD8+ T cells, NK cells, NKT cells, which play roles in the development of diseases.We investigated Tim-3 expression on NK cells and NKT cells from peripheral blood of 79 lung cancer cases and 53 healthy controls.The gating strategy to distinguish the various lymphocyte populations is shown in Figure 1A.Increased proportion of Tim-3+ cells was detected on CD3-CD56+ cells in lung cancer patients than in healthy controls (73.8%±9.01%vs 70.4%±7.47%,p=0.03, Figure 1B).When NK cells were subtyped based on CD56 expression levels, the proportion of Tim-3+ cells in CD3-CD56 dim cells was elevated in lung cancer cases than in controls (75.3%±7.63%vs 72.4%±7.68%,p=0.03, Figure 1C).However, Tim-3 expression was not increased in CD3-CD56 bright cells in cases compared to controls (58.8% ± 13.1% vs 58.2% ± 6.43%, p=0.91, Figure 1D).Additionally, the proportion of Tim-3+ cells  in CD3+CD56+ cells was also significantly increased in cases compared to controls (51.6%±14.1% vs 47.0%±8.32%,p=0.04, Figure 1E).These results indicate that Tim-3 may be involved in the pathogenesis of lung cancer by its regulation on various immune cells.

Effects of surgical resection on Tim-3 expression of CD3-CD56+ NK cells and CD3+ CD56+ NKT cells in patients with lung cancer
We examined Tim-3 expression in postsurgical blood samples from 21 of the patients with lung cancers in our cohort within 4 to 20 weeks after surgery, including 8 cases with stage I/II and 13 cases with stage III/IV, which stage were assessed when patients were in hospital and underwent surgery.The results showed that Tim-3 expression on CD3-CD56+ cells, CD3-CD56 dim cells, and CD3+CD56+ cells after surgery was significantly lower than those before surgery (CD3-CD56+ cells, p﹤0.01, Figure 3A; CD3-CD56 dim cells, p﹤0.01, Figure 3B; CD3+CD56+ cells, P ﹤ 0.01, Figure 3D).However, there was no difference in Tim-3 expression of CD3-CD56 bright cells before and after surgery of lung cancer (p=0.78, Figure 3C).Our data suggest that Tim-3-expressing immune cells are far more prevalent before resection of the primary tumor.

Discussion
Many reports have indicated that the dysfunction of NK cell and NKT cell is closely related to the occurrence and development of lung cancer (Hasegawa et al., 2014;Hodge et al., 2014), but the underlying molecular mechanisms involved in this process remain poorly understood.Tim-3, was originally identified for Th1specific markers.Since then, it was also been found on CD8+ T cells, Treg, monocytes, dendritic cells, NK cells and NKT cells.Here, to our knowledge for the first time, we demonstrated that Tim-3 expression was significantly increased on CD3-CD56+ NK cells, CD3-CD56 dim NK cells and CD3+CD56+ NKT cells in lung cancer patients.In addition, Tim-3 expressions on these cells in cases with high stages (III/IV) were higher than those with low stages (I/II).More importantly, we found that Tim-3 expression on these cells after surgery was significantly lower than those before surgery.These suggest that Tim-3 could be a valuable marker for lung cancer.Further investigations with a larger scale of lung cancer from blood samples and the 5-year survival rate of patients are needed to establish to further confirm the role of Tim-3 as a potential diagnostic marker and a therapeutic target for the treatment of lung cancer.
It has been reported that Tim-3 has a negative regulatory role on the activity of NK cells, and chronic hepatitis B infection could upregulate Tim-3 expression on NK cells and subsequently suppressed NK cell function (Ju et al., 2010).It was also demonstrated that Tim-3  suppressed NK cell-mediated cytotoxicity when Tim-3 encountered target cells (Ndhlovu et al., 2012).NK cells in non-small cell lung cancer (NSCLC) patients is significantly lower than that of healthy controls (Wang et al., 2013), suggesting NK cell function was damaged in patients with lung cancer.Our data showed that Tim-3 expressions in patients with lung cancer were elevated on CD3-CD56+ NK cells, which might be an underlying mechanism for dysfunction of NK cells in lung cancer.When NK cells were subtyped based on CD56 expression levels, we found that Tim-3 expressions in patients with lung cancer were elevated on CD3-CD56 dim NK cells while Tim-3 expressions were not changed on CD3-CD56 bright NK cells.About 90% of human NK cells express low density CD56 (CD56 dim ), which can effectively exert cytotoxicity, and about 10% NK cell phenotypes are CD56 bright, which function is to produce cytokines.CD56 bright NK cells may differentiate into CD3-CD56 dim NK cells over time, and recent data have shown that Tim-3 acts as an NK cell maturation marker (Ndhlovu et al., 2012) and is necessary for their cytotoxicity, which could explain the results in our study.Additionally, our data showed that Tim-3 expression was significantly increased on CD3+CD56+ NKT cells in lung cancer patients.Liu Y et al reported that there was a negative correlation between Tim-3 expression and TNF-α production by NKT cells (Liu et al., 2010), suggesting that Tim-3 may also play a negative role in regulating NKT cell function, and it was also consistent with our results.Cao X et al demonstrated that Tim-3 is highly upregulated on both CD4+ and CD8+ tumor-infiltrating lymphocytes (TILs) from human lung cancer tissues (Cao et al., 2012), which also supports our view on this, the increased Tim-3 expression may involved in the immunological dysfunctions of lung cancer.
Lung cancer is categorized by stages, depending on how far they have spread beyond the lung.The higher the stage number, the worse the situation.Our data showed that there was a positive correlation between disease stages and Tim-3 expression on CD3-CD56+ NK cells, CD3-CD56 dim NK cells and CD3+CD56+ NKT cells.It is the first report Tim-3 expression on immune cells was associated with stages of lung cancer patients.Lung cancers are divided into stage I, II, III, IV and high stage means the cancer is more serious and the disorder of immune cells is more serious.Our data demonstrated that cases with stages III/IV had significantly higher proportion of Tim-3+ CD3-CD56+ NK cells, Tim-3+CD3-CD56 dim NK cells and Tim-3+CD3+CD56+ NKT cells than those with stages I/II.These results suggested that Tim-3 on these immune cells may play roles in the progression of lung cancer.In addition, we show that surgical resection of primary tumor rapidly reverses Tim-3 expression on CD3-CD56+, CD3-CD56 dim and CD3+CD56+ populations, which has significant implications for the timing of Tim-3-based therapies, and also suggesting Tim-3 blocking therapies could be more effective if started before surgery.
In summary, the expression of Tim-3 is upregulated on NK and NKT cells from peripheral blood in lung cancer, and is positively correlated with stages.In addition, Tim-3 expression on NK and NKT cells is reduced after surgical resection.Our data suggest that Tim-3 might be a potential diagnostic marker for the progression of lung cancer and might be as a therapeutic target for the treatment of this disease.

Figure 1 .
Figure 1.Tim-3 Expression on Peripheral CD3-CD56+ NK Cells and CD3+CD56+ NKT Cells in Lung Cancer.A) Representative dot plots of peripheral blood from one patient showing the gating strategy for the various lymphocyte populations; B, C, D, E) show the percentage of Tim-3+CD3-CD56+ cells, Tim-3+CD3-CD56 dim cells, Tim-3+CD3-CD56 bright cells and Tim-3+CD3+CD56+ cells (means ± SD) compared between lung cancer patients and healthy controls, respectively.Each dot represents one subject.The student t-test was used for comparison between groups.P-Values are shown.Value of p<0.05 is considered as a significant difference

Figure 2 .
Figure 2. Comparison of Tim-3 Expressions on CD3-CD56+ Cells (A), CD3-CD56 dim Cells (B), CD3-CD56 bright Cells (C) and CD3+CD56+ cells (D) in Patients with Different Disease Stages.79 patients were included, in which 37 patients were in stages I or II, and 42 patients were in stages III or IV.The student t-test was used for comparison.P-Values are shown.Value of p﹤0.05 is considered as a significant difference Figure 2

Table 1 . General Characteristics of the Subjects
*Statistical comparisons were made by using the Student's t-test.Differences were considered significant when the value of P was equal to or less than 0.05.NS = not statistically significant.Ad, Adenocarcinoma; Sq, Squamous cell carcinoma