| Objective: To investigate the levels of T cell and special CD4+CD25+ Regulatory T cells (Tregs) in peripheral blood of the patients with gastric cancer, and to explore the possible function and mechanism of these Tregs in suppressing anti-tumor response of T cells (Cytotoxic T Lymphocyte , CTLs) and antigen presenting of dendritic cells (DCs) in adoptive immunotherapy of gastric carcinoma. Furthermore, in order to understand the possible function of Tregs in regulating anti-cancer response of the patients with gastric carcinoma, the expression of FoxP3 gene in these Treg cells was further characterized.Methods: By using flow cytometry, the levels of CD3+T cells, CD8+T cells, and CD4+CD25+T cells in peripheral blood of preoperative patients with gastric cancer were detected respectively, and the results were analyzed statistically in comparison with that of the healthy volunteers. The Dendritic cells separated from peripheral blood of the patients with gastric cancer and the healthy volunteers were activated by culturing in vitro. Expression of CD80 and CD83 on the surface of mature dendritic cells was detected by flow cytometry. The immunocytes separated from peripheral blood of the patients with gastriccancer were activated into antigen specific CTLs by mature DCs loaded with tumor antigen and proliferated in vitro. Cytotoxicity assay against gastric cancer cell line named BGC-823 cells was performed by using CCK-8 Kit with the E/T ratio of 20:1. RT-PCR techniques were used to detect the expression of FoxP3 gene in these Treg cells, and the results were analyzed statistically in comparison with that in the healthy volunteers.Results:1 Percentages of CD4+CD25+T cells in patients and volunteers: The percentage of total T lymphocytes (CD3+T) and inhibiting killer T cells (CD8+T) in peripheral blood of the preoperative patients with gastric cancer was found to be no statistical difference in comparison with that of the healthy volunteers. The level of induced helper T cells (CD4+T) in the patients with gastric cancer was lower than that of volunteers (t=2.636, P<0.05), while the percentage of CD4+CD25+T cells in total CD4+T cells in the patients with gastric cancer went up significantly (12.76±0.46)% in comparison with that in the healthy volunteers [(6.81±0.66)%; t=7.441, P<0.01].2 Expression of CD80 and CD83 on mature dendritic cells in patients and volunteers: The expression of CD80 and CD83 on the surface of mature dendritic cells of the patients with gastric cancer were (59.95±1.01)% and (48.66±1.27)%, and that of healthy volunteers were (75.04±0.60)% and (75.68±0.58)%, respectively. The expressions of CD80 and CD83 in the patients with gastric cancer were significantly lower than that in the healthy volunteers (tCD80=12.90,P<0.01; tCD83=19.43,P<0.01).3 The association of the level of Tregs and the expression of DC markers in patients: The percentage of Tregs in peripheral blood of the patients with gastric cancer were significantly increased accompanied with reduced expression of CD80 on the surface of mature dendritic cells in the patients with gastric cancer. The same situation happened to CD86. A negative correlation was found in the level of Tregs and the expression of DC markers in patients with gastric cancer (r CD80=0.603,P<0.01; r CD83=0.463,P<0.05).4 Anti-tumor responses in cytotoxicity assay: The specific cytotoxicity in experimental group in which CD4+CD25+T cells were removed from PBMCs(28.45±2.89)% was significantly higher than that in control group with CD4+CD25+T cells non-removed〔(16.29±3.04)%;t=2.898,P<0.01〕.5 Expression of FoxP3 gene in Tregs of the patients: The expression of FoxP3 gene in Tregs was significantly increased in the patients with gastric cancer〔(0.84±0.02)%〕in comparison with that in the healthy volunteers〔(0.76±0.03)%; t=2.229, P<0.05〕.Conclusions:1. Decreased T effector cells and significantly increased Tregs in peripheral blood of the patients with gastric cancer indicated that Tregs might play a pivotal role in the processes of self-tumor immunity of the patients with gastric cancer.2. Reduced expression of CD80 and CD86 on the surface of mature dendritic cells of the patients with gastric cancer and its negative correlation with the level of Tregs in peripheral blood demonstrated that Treg might simultaneousely participate in the regulating of DC functions.3. Cytotoxicity of antigen specific CTLs could be significantly enhanced when Tregs were removed confirmed that Tregs might directly or indirectly play a crucial role in regulating the self-tumor immune-surveillence and immune-tolerance of the patients with gastric cancer.4. Increased FoxP3 gene expression of Treg cells indicated that FoxP3 gene might associated with the regulatory/suppressive function of the Tregs in self-tumor immunity of the patients with gastric cancer.Present study suggests that an effective cancer immunotherapy might be come true by adjusting and controlling the levels of Tregs or down regulating the expression of FoxP3 gene.
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