Sun et al. Journal of Experimental & Clinical Cancer Research 2011, 30:82 http://www.jeccr.com/content/30/1/82 Page 5 of 10 apoptotic compared with 59.80 ± 11.46% of CD3+ T cells co-cultured with CHO/EGFP cells, and 32.40 ± 6.40% of CD3+ T cells that were cultured alone. The differences were statistically significant (P < 0.05), indi- cating that IDO+ CHO cells could induce significant T cell apoptosis. Furthermore, after added the 1-MT, the specific inhibitor of IDO in co-culture of CD3+T cells and IDO+ CHO cells, the apoptosis could not be induced (only 33.1 ± 4.87% of CD3+T cells were apop- totic) (Figure 2). In vitro induction of peripheral CD4 + CD25 + CD127- T cells by IDO+ CHO cells in the peripheral blood of breast cancer patients Mononuclear cells isolated from the peripheral blood of breast cancer patients were incubated with IDO+ CHO cells to assess the effect of IDO expression on Treg cells. After 7 days of incubation of 2 × 106 CD3+ T cells in media containing 200 U/ml IL-2, CD4+CD25 +CD127- Tregs were 3.43 ± 1.07% of the CD3+T cell population. However, after 7 days of co-culture of 1 × 105 CHO cells expressing IDO or EGFP and 2 × 106 CD3+ T cells, CD4+CD25+CD127- Tregs were 8.98 ± 1.88% of the CD3+T cell population in co-cultures with IDO+ CHO cells, but were only 3.73 ± 1.12% of the CD3+T cell population in co-cultures with CHO/EGFP cells (Figure 3). The proportion of Tregs in co-cultures of CD3+ T cells and IDO+ CHO cells was higher than in the other two groups, and the differences were statis- tically significant (P < 0.05). After added the inhibitor 1- MT, CD4+CD25+CD127-Tregs were 5.1 ± 1.30% of the CD3+T cell population in co-cultures with IDO+ CHO cells. It confirmed that the IDO had the function to induce the peripheral Tregs. RT-PCR analysis of Foxp3 gene expression Seven days following co-culture of IDO+ CHO cells and CD3+ T cells, Foxp3 gene expression was detected in the CD3+ T cells by RT-PCR analysis. CD3+T cells alone and CD3+T cells co-cultured with CHO/EGFP cells were used as negative controls. The value of the Foxp3 and b-actin gray scale ratios in CD3+ T cells co- cultured with IDO+ CHO cells, CD3+ T cells and CD3+ T cells co-cultured with CHO/EGFP cells were 0.5567 ± 0.1271, 0.3283 ± 0.1530 and 0.3800 ± 0.0748, respectively. The value of the Foxp3 and b-actin gray scale ratio in the T cells co-cultured with IDO+ CHO cells was higher than in the control groups (P < 0.05) (Figure 4A). Quantitative real-time RT-PCR analysis of Foxp3 gene expression Foxp3 gene expression was detected in CD3+T cells after 7 days of co-culture with IDO+ CHO cells by quantitative RT-PCR analysis. CD3+T cells and CD3+T cells co-cultured with CHO/EGFP cells were used as negative controls. The relative expression of Foxp3 in CD3+ T cells from IDO+ CHO cell co-cultures, in CD3 + T cells and in CD3+T cells from co-cultures with CHO/EGFP cells were 0.00056 ± 0.00012, 0.00028 ± 0.00013 and 0.00023 ± 0.00005, respectively. Relative Foxp3 gene expression was higher in T cells co-cultured with IDO+ CHO cells than in T cells from the control groups (P < 0.05) (Figure 4B). Western blot analysis of Foxp3 expression Foxp3 protein expression was detected in CD3+ T cells 7 days after co-culture with IDO+ CHO cells. CD3+T cells and CD3+T cells co-cultured with CHO/EGFP cells were used as negative controls. Cell lysates from T cells isolated from co-cultures with IDO+ CHO cells contained a 48 kDa protein band reactive to a Foxp3- specific monoclonal antibody. This band was not pre- sent in cell lysates from T cells from the control group cultures (Figure 4C). Discussion IDO is expressed in many human and animal tissues and cells as well as on the surface of human tumor cells. An in-depth analysis is needed to identify the spe- cific mechanisms that underly the role of IDO in tumor immune tolerance. Recent studies have shown that acute myeloid leukemia (AML) cells that express IDO can transform CD4+CD25-T cells into CD4+CD25+T cells [12]. However further study is needed to elucidate the mechanism behind this transformation and the rela- tionship between IDO and Treg cells in solid tumors [13-18]. In this study, we constructed a stable cell line expressing IDO and carried out preliminary in vitro ana- lysis of the induction effect of IDO on Tregs isolated from the peripheral blood of patients with breast cancer. IDO is expressed both in tissues of patients with breast cancer and in breast cancer cell lines [19,20]. In this study, during the preparation of the IDO gene expression vector, we identified IDO gene expression in the human breast cancer cell lines MDA-MB-231, MDA-MB-435S, MDA- MB-453, SK-Br-3, T47D, ZR-75-1 and normal breast cells HBL-60; the gene was highly expressed in MDA-MB- 435S, T47D, MCF-7. We also detected IDO expression in patients with primary breast cancer and in lymph nodes draining the tumor; IDO expression in lymph node tissue was consistent with results previously reported in the lit- erature [4,21,22]. Moreover, in our previous study, we found that the proportion of CD4+CD25+ regulatory T cells in the peripheral blood of patients with breast cancer was higher than that in the peripheral blood of patients with benign breast tumors and healthy volunteers; the pro- portion of CD4+CD25+Tcells was directly related to tumor size [23]. This phenomenon suggests that in