Immunological milieu in the peritoneal cavity at laparotomy for gastric cancer

Abstract

Aim: To investigate the immunological repertoire in the peritoneal cavity of gastric cancer patients.

Methods: The peritoneal cavity is a compartment in which immunological host-tumor interactions can occur. However, the role of lymphocytes in the peritoneal cavity of gastric cancer patients is unclear. We observed 64 patients who underwent gastrectomy for gastric cancer and 11 patients who underwent laparoscopic cholecystectomy for gallstones and acted as controls. Lymphocytes isolated from both peripheral blood and peritoneal lavage were analyzed for surface markers of lymphocytes and their cytokine production by flow cytometry. CD4(+)CD25(high) T cells isolated from the patient's peripheral blood were co-cultivated for 4 d with the intra-peritoneal lymphocytes, and a cytokine assay was performed.

Results: At gastrectomy, CCR7(-) CD45RA(-) CD8(+) effector memory T cells were observed in the peritoneal cavity. The frequency of CD4(+) CD25 (high) T cells in both the peripheral blood and peritoneal cavity was elevated in patients at advanced stage [control vs stage IV in the peripheral blood: 6.89 (3.39-10.4) vs 15.34 (11.37-19.31), P < 0.05, control vs stage IV in the peritoneal cavity: 8.65 (5.28-12.0) vs 19.56 (14.81-24.32), P < 0.05]. On the other hand, the suppression was restored with CD4(+) CD25(high) T cells from their own peripheral blood. This study is the first to analyze lymphocyte and cytokine production in the peritoneal cavity in patients with gastric cancer. Immune regulation at advanced stage is reversible at the point of gastrectomy.

Conclusion: The immunological milieu in the peritoneal cavity of patients with advanced gastric cancer elicited a Th2 response even at gastrectomy, but this response was reversible.

Keywords: Cytokines; Gastric cancer; Lymphocytes; Peritoneal cavity; Regulatory T cell.

Figure 1

Co-staining with foxp3 and CD25 for CD4⁺ T cells. High correlation was shown between both populations.

Figure 2

Analysis of lymphocyte populations in peripheral blood and the peritoneal cavity. A: The gating and counting of CD4⁺ CD25^high T cell population by flow cytometry; B: The percentage of CD4⁺ CD25^high T cells in the CD4⁺ T cell population in peripheral blood and peritoneal lavage of patients at each stage of gastric cancer and control patients. Data are presented as the mean ± SD.

Figure 3

Cytokine production by lymphocytes. A: The gating and counting of the IFN-γ producing cell population by flow cytometry; B: The percentage of IFN-γ producing cells in the CD3⁺ cell population stimulated with PMA + ionomycin in peripheral blood and peritoneal lavage of patients at each stage of gastric cancer and control patients Data are presented as the mean ± SD. The statistical analysis was performed by the Kruskal-Wallis test. After gating of CD3+ T cells, 10  000 events were analyzed. The production of IFN-γ in the peritoneal cavity was higher than that in the peripheral blood. The ratio of IFN-γ production cells in the peritoneal lavage was significantly lower in patients with advanced-stage than in controls [control vs stage IV: 51.1 (35.1-67.1) vs 10.7 (2.6-22.1), ^aP < 0.05]; C: The gating and counting of the IL-10 producing cell population by flow cytometry; D: The percentage of IL-10 producing cells in the CD3⁺ cells stimulated with PMA + ionomycin in peripheral blood and peritoneal lavage of patients at each stage of gastric cancer and control patients. Data are presented as the mean ± SD. The ratio of IL-10 producing cells in peripheral blood and intra-peritoneal lymphocytes was significantly higher in patients at advanced stage than in controls [control vs stage IV: 6.1 (3.94-8.25) vs 40.7 (18.35-63.0), ^aP < 0.05].

Figure 4

Cytokine assays of intra-peritoneal lymphocytes after co-cultivation with self-CD4⁺CD25 ^highT cells. A: IFN-γ production in intra-peritoneal lymphocytes co-cultivated with self- CD4⁺ CD25^high T cells; B: Either CD4⁺ CD25^high T cells or CD4⁺ CD25^- T cells.