Detection and significance of TregFoxP3(+) and Th17 cells in peripheral blood of non-small cell lung cancer patients

Abstract

Introduction: The aim of this study was to explore the relationships between TregFoxP3(+) cells and Th17 cells and occurrence of lung cancer.

Material and methods: The proportions of TregFoxP3(+) and Th17 cells, the expression of FoxP3 and RORγt mRNA, and the levels of related cell factors such as transforming growth factor-β (TGF-β), interleukin IL-17 (IL-17) and IL-23 were determined respectively by flow cytometry analysis, real-time-polymerase chain reaction (PCR), and ELISA in peripheral blood of 18 healthy people and 26 patients with non-small cell lung cancer (NSCLC).

Results: The levels of TregFoxP3(+) and Th17, expression of FoxP3 and RORγt mRNA, and ratios of TregFoxP3(+)/Th17 and FoxP3/RORγt in peripheral blood with NSCLC were higher than those in healthy controls (p < 0.05). The proportion of Th17 cells from NSCLC patients was positively correlated with that of TregFoxP3(+) (r = 0.81, p < 0.05). The receiver-operating characteristic (ROC) curve demonstrates that the increased level of TregFoxP3(+)/Th17 in the peripheral blood may be a useful indicator in early diagnosis of non-small cell lung carcinoma. The TregFoxP3(+)/Th17 and FoxP3/RORγt levels for patients in stage IV were higher than those of patients in stages I, II, and III (p < 0.05). The levels of TGF-β, IL-17, and IL-23 were higher in NSCLC patients than those in healthy controls.

Conclusions: The results suggest that ratios of Treg/Th17 correlate with the stage of NSCLC.

Keywords: RORγt; Th17; TregFoxP3+; carcinoma; non-small cell lung.

Figure 1

Proportions of TregFoxP3⁺ in the healthy control group (A) and the NSCLC group (B). Isolated PBMCs were labeled by anti-CD4, anti-CD25 and anti-FoxP3 antibodies before cytometric analysis. Representative flow cytometric results are shown. The cells positive for CD4 and FoxP3 were gated (upper right quadrant). Experiments were conducted three times. A p value of less than 0.05 was considered statistically significant

Figure 2

Proportions of Th17 in the healthy control group (A) and the NSCLC group (B). Isolated PBMCs were labeled by anti-CD4 and anti-Th17A antibodies before cytometric analysis. Representative flow cytometric results are shown. The cells positive for CD4 and Th17A were gated (upper right quadrant). Experiments were conducted three times. A p value of less than 0.05 was considered statistically significant

Figure 3

TregFoxP3 ⁺ and Th17 correlation analysis in NSCLC patients. The linear correlation method was used. The sign and the absolute value of a correlation coefficient describe the direction and the magnitude of the relationship between two variables. The value of a correlation coefficient ranges between –1 and 1. The greater the absolute value of a correlation coefficient, the stronger the linear relationship. In our study the correlation coefficient (r) between TregFoxP3 ⁺ and Th17 was 0.81

Figure 4

Receiver-operating characteristic (ROC) curve analysis to assess the performance of Treg- FoxP3⁺/Th17 in the diagnosis of non-small cell lung carcinoma. The TregFoxP3⁺/Th17 ratio was used as an indicator variable. The ROC curve was constructed by plotting the true positive rate (sensitivity) and false positive rate (specificity) associated with each unique value of TregFoxP3⁺/Th17 ratio. An indicator variable with high discriminatory ability will have a curve with an AUC near 1, and an indicator variable with low discriminatory ability will have an AUC near 0.5. In this study, the AUC was 0.809

Figure 5

TregFoxP3⁺/Th17 among different TNM stages in NSCLC patients. The non-parametric rank sum test was conducted. According to TNM staging, 6 patients in stage I–II, 12 patients in stage III and 8 patients in stage IV were enrolled in this analysis. Experiments were performed three times. A p value of less than 0.05 was considered statistically significant

Figure 6

FoxP3/RORγt among different TNM stages in NSCLC patients. The non-parametric rank sum test was conducted. According to TNM staging, 6 patients in stage I–II, 12 patients in stage III and 8 patients in stage IV were enrolled in this analysis. Experiments were performed three times. A p value of less than 0.05 was considered statistically significant

Figure 7

The level of TGF-β, IL-17 and IL-23 in the serum of the healthy control group and the NSCLC group ($\overline{x}$ ± s, pg/ml). ELISA assay was performed to detect the expression levels of TGF-β, IL-17 and IL-23. Experiments were performed three times and the data were expressed as $\overline{x}$ ± s. A p value of less than 0.05 was considered statistically significant

Figure 8

The ratio of TGF-β, IL-17 and IL-23 in the early-mid group/healthy control group and the advanced stage group/healthy group. The relative level of TGF-β, IL-17 and IL-23 was compared between the early-mid group and the healthy control group; and between the advanced stage group and the healthy group. Experiments were performed three times. A p value of less than 0.05 was considered statistically significant