Distribution of Th17 cells and FoxP3(+) regulatory T cells in tumor-infiltrating lymphocytes, tumor-draining lymph nodes and peripheral blood lymphocytes in patients with gastric cancer

Abstract

Although Th17 cells reportedly play critical roles in the development of autoimmunity and allergic reactions, information on Th17 cells in cancer-bearing hosts is still limited. In the present study, we investigated the distribution of Th17 cells in relation to regulatory T cells (Treg) in the tumor-infiltrating lymphocytes (TILs), regional lymph node lymphocytes, and peripheral blood lymphocytes of gastric cancer patients. Interleukin (IL)-17-producing CD4(+) cells as Th17 cells and CD4(+)CD25(+)FoxP3(+) cells as Treg were evaluated by flow cytometry and expressed as a percentage of the total CD4(+) cells, in addition to performing a Th1/Th2 balance assay. Moreover, immunohistochemical staining for IL-17 and FoxP3 were performed. In TILs from patients with early disease (n = 27), the frequency of Th17 cells was significantly higher than that in the normal gastric mucosa (23.7 ± 8.9 vs 4.5 ± 3.1%). In TILs from patients with advanced disease (n = 28), the frequency of Th17 cells was also significantly higher, but lower compared to early disease, than that in the normal gastric mucosa (15.1 ± 6.2 vs 4.0 ± 2.0%). This observation for Th17 cell-distribution was also confirmed by immunohistochemistry. When the ratio of Th17/Treg in TILs was evaluated in individual cases, it was more markedly increased in early than in advanced disease. In conclusion, the accumulation of Th17 cells as well as Treg in the tumor microenvironment of gastric cancer occurred in early disease and then the infiltration of Th17 cells gradually decreased according to the disease progression, in contrast to increased Treg.

Figure 1

Prevalence of Th17 cells in gastric cancer. (a) Representative flow cytometric data showing interleukin (IL)‐17‐producing CD4(+) cells. (b) The frequencies of Th17 cells (%Th17) in tumor‐infiltrating lymphocytes (TILs) in comparison to intra‐epithelial lymphocytes (IELs) of normal gastric mucosa are shown. The patients were divided into early disease (early) corresponding to stage I and advanced disease (advanced) corresponding to stages II, III, and IV. (c) The frequencies of Th17 cells (%Th17) in peripheral blood mononuclear cells (PBMCs) in comparison with healthy donors (Healthy). (d) Regional lymph nodes in the stomach were classified into N1 regional lymph nodes adjacent to the gastric tumor and N2 regional lymph nodes marginally distant from the tumor, according to the Japanese Classification of Gastric Carcinoma. Mesenteric lymph nodes were used as control lymph nodes (Nc).

Figure 2

Immunohistochemical staining for inter‐leukin (IL)‐17 in gastric cancer. Representative immunostaining for IL‐17 is shown for advanced and early gastric cancer in addition to normal gastric mucosa and negative control. Summarized data with quantitative analysis indicated that the frequency of Th17 cells in tumor‐infiltrating lymphocytes (TILs) from patients with advanced disease was significantly decreased compared to that in those with early disease.

Figure 3

Prevalence of regulatory T cells in gastric cancer. (a) Representative flow cytometric data showing CD4(+)CD25(+)FoxP3(+) cells. (b) The frequencies of regulatory T cells (%FoxP3) in tumor‐infiltrating lymphocytes (TILs) in comparison to intra‐epithelial lymphocytes (IELs) of normal gastric mucosa are shown. (c) The frequencies of regulatory T cells (%FoxP3) in peripheral blood mononuclear cells (PBMCs) in comparison with healthy donors (healthy). (d) Prevalence of regulatory T cells in lymph nodes of gastric cancer.

Figure 4

Immunohistochemical staining for FoxP3(+) regulatory T cells in gastric cancer. Representative immuno‐staining for FoxP3 were shown in advanced and early stage of gastric cancer in addition to normal gastric mucosa and negative control.

Figure 5

Balance of Th17/regulatory T cells (Treg) according to the progression of gastric cancer. (a) The ratio of Th17/Treg was evaluated in individual cases. (b) Each value of the prevalence of Th17 (%Th17) and regulatory T cells (%FoxP3) in tumor‐infiltrating lymphocytes (TILs) was plotted.

Figure 6

Cytokine productions of T cells. Supernatant produced by peripheral blood mononuclear cells (PBMCs) or N1 regional lymph node lymphocytes in response to CD3 plus CD28 stimulation was evaluated using ELISA for interleukin (IL)‐17, IL‐21, and IL‐23. In Figure 6(a,b), the levels of IL‐17 production were significantly correlated with the prevalence of Th17 cells evaluated by FACS analysis.