CD4(+)CD25high regulatory T cells increase with tumor stage in patients with gastric and esophageal cancers

Abstract

Purpose: Regulatory T cells (T regs) can inhibit immune responses mediated by T cells. It has been shown that there is an increased proportion of T regs in several different human malignancies, although the actual mechanism remains unclear. In the present study, we evaluated the prevalence of CD4(+)CD25high T regs in PBMCs from patients with gastric and esophageal cancers in relation to the clinical outcome.

Methods: PBMCs in 72 patients with gastric cancer and 42 patients with esophageal cancer were evaluated for the proportion of CD4(+)CD25high T cells, as a percentage of the total CD4(+) cells, by flow cytometric analysis with triple-color staining. Actuarial overall survival rates of the patients were analyzed by the Kaplan-Meier method.

Results: The percentages of CD4(+)CD25high T cells for cases of gastric cancer (4.9+/-1.2%) and esophageal cancer (5.2+/-2.1%) were significantly higher than those for healthy donors (1.9+/-1.1%, P<0.01). There were significant differences in the prevalence of CD4(+)CD25high T cells between the early and advanced disease stages, both in gastric cancer (stage I vs. III, P<0.05; stage I vs. IV, P<0.05) and esophageal cancer (stage I vs. IV, P<0.05). The patients with a high proportion of CD4(+)CD25high T cells showed poorer survival rates in comparison to those with a low proportion, in both gastric and esophageal cancers. After patients received curative resections of gastric cancers (n=57), the increased proportions of CD4(+)CD25high T cells were significantly reduced, and the levels were almost equal to those in normal healthy donors. In addition, studies of gastric cancer patients with postoperative recurrent tumors (n=6) revealed that the prevalence of CD4(+)CD25high T cells individually increased compared to 2 months after the operations. CD4(+)CD25high T cells expressed FOXP3 mRNA and had abundant CD45RO and intracellular CTLA-4 molecules.

Conclusions: These results strongly suggest that tumor-related factors induce and expand CD4(+)CD25high T regs.

Fig. 1

CD4(+)CD25^high T cells in PBMCs from patients with gastric and esophageal cancers. The population of CD4(+)CD25^high T cells as a percentage of the total CD4(+) cells was evaluated by flow cytometric analysis with triple-color staining. Representative flow cytometric data from gastric (stage III) and esophageal cancer patients (stage III) and healthy donors are shown. Rectangular gates indicate CD4(+)CD25^high T cell populations

Fig. 2

Increased populations of CD4(+)CD25^high T cells in PBMCs from patients with gastric and esophageal cancers. The percentages of CD4(+)CD25^high T cells in cases of gastric (n=72, 4.9±1.2%) and esophageal cancers (n=42, 5.2±2.1%) were significantly higher than those of healthy donors (n=16, 1.9±1.1%, P<0.01). There were significant differences in the prevalence of CD4(+)CD25^high T cells between stage I and III (P<0.05), stage I and IV (P<0.05) in gastric cancer, as well as stage I and IV (P<0.05) in esophageal cancer. Stage classification was defined according to the TNM classification

Fig. 3

The expression of CD45RO and intracellular CTLA-4(CD152) on CD4(+)CD25^high T cells. Representative flow cytometric data from gastric cancer patients (stage III) showed the expression of CD45RO and intracellular CTLA-4 after the gating of CD4(+)CD25^high T cells

Fig. 4

Intracellular cytokine staining of CD4(+)CD25^high T cells. Intracellular cytokine staining (IFN-γ and IL-10) was performed gated on CD4(+)CD25^high T cells (a) or CD4(+)CD25^int T cells (b) derived from the PBMCs in the gastric cancer patient (stage III)

Fig. 5

FOXP3 mRNA analysis of CD4(+)CD25^high T cells. CD4(+)CD25^high T regs were separated by FACS sorting on CD4(+)CD25^high T cells in PBMCs from gastric cancer patients (stage III and IV). Total RNA was extracted from sorted CD4(+)CD25^high T cells (samples 1–4) or CD4(+)CD25int T cells (samples 5, 6) and RT-PCR analyze specific for FOXP3 mRNA was performed

Fig. 6

Survival rates of patients in relation to the prevalence of CD4(+)CD25^high T cells. PBMCs were separated prior to the operation and analyzed for the prevalence of CD4(+)CD25^high T cells. When the patients were separated into high or low prevalence of T regs groups, classified by the mean values of CD4(+)CD25^high T cells, gastric cancer patients with high CD4(+)CD25^high T cell levels (n=38) showed poorer survival rates in comparison to those with low CD4(+)CD25^high T cell levels (n=34), analyzed by the log-rank test (a). Similarly, esophageal cancer patients with high CD4(+)CD25^high T cell levels (n=22) showed poorer survival rates in comparison to those with low CD4(+)CD25^high T cell levels (n=20) (b)

Fig. 7

Clinical course of patients in relation to the prevalence of CD4(+)CD25^high T cells. The prevalence of CD4(+)CD25^high T cells between preoperative and postoperative (2 months after the operation) periods are shown in a. After patients had received curative resections of gastric cancers (n=57), the increased levels of CD4(+)CD25^high T cells were significantly reduced, analyzed by the paired t test (a). In addition, studies for gastric cancer patients with postoperative recurrent tumors (n=6, postrecurrence) showed that the prevalence of CD4(+)CD25^high T cells individually increased in comparison to 2 months after the operation (prerecurrence) (b)