Sun et al. Journal of Experimental & Clinical Cancer Research 2011, 30:82 http://www.jeccr.com/content/30/1/82 Page 2 of 10 Introduction The molecular mechanisms underlying tumor-induced tolerance are the subject of active research, and a num- ber of contributing mechanisms have been identified. Indoleamine 2, 3-dioxygenase (IDO/INDO), an impor- tant enzyme in the metabolism of tryptophan, catalyzes the rate-limiting step of tryptophan degradation along the kynurenine pathway. Reduction in the local trypto- phan concentration and generation of tryptophan meta- bolites can suppress T cell proliferation or induce T cell apoptosis [1,2], and IDO has been implicated in the endogenous induction of peripheral tolerance and immunosuppression [3,4]. In addition, many human solid tumors express IDO, indicating that it may contri- bute to the induction of tumor tolerance [5-8]. Regulatory T cells (Tregs [CD4+CD25+CD127-]) can inhibit most types of immune responses and are emer- ging as a key component of acquired tolerance to tumors [9]. Increased Treg activity facilitates tumor growth, whereas depletion of Tregs allows for effective anti-tumor immune responses [10]. Previous studies have shown that IDO is expressed in tumor-draining lymph nodes. Interestingly, we previously found that IDO expression in primary breast cancer tumors is accompanied by Treg infiltration (unpublished data), suggesting a correlation between IDO activity and Tregs in these tumors. However, the role of increased IDO expression in tumor cells in development of Treg cells is not clear. In this study, we investigated the potential effects of IDO on development of Treg cells in breast cancer tumors using a stable IDO-expressing Chinese hamster ovary (CHO) cell line. Materials and methods Cell lines and culture conditions The Chinese hamster ovary (CHO) cell line was pur- chased from the Shanghai Institute of Cell Biology, Chi- nese Academy of Sciences (Shanghai, China). The breast cancer cell line MDA-MB-435s was obtained from American Type Culture Collection (Manassas, VA). Both cell lines were maintained in culture as adherent monolayer in RPMI-1640 (Gibco, Invitrogen Corp., Carlsbad, CA) medium supplemented with 10% fetal bovine serum (FBS), L-glutamine (1%) and penicillin (0.1%). Cells were incubated at 37°C in a humidified atmosphere with 5% CO2. Construction of a recombinant plasmid containing human IDO cDNA Total RNA was isolated from breast cancer MDA-MB- 435s cells using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. A 1225 kb fragment encompassing the entire coding region of human IDO cDNA was obtained using RT-PCR (Takara, Dalian, China) with the following primer pair: sense 5’- AGATCTGCCACCATGGCACACGCTATGGAAAAC- 3’, and antisense 5’-GTCGACTTAACCTTCCTT- CAAAAGGGATTTC-3’. The PCR products were inserted into the pMD19-T Simple Vector (Takara) using TA-cloning procedures, and sequencing analysis was used to identify the product of interest (pMD19- IDO). Establishment of stable transformants For construction of stable transformants, pMD19-IDO and pIRES2-EGFP (Clontech, Santa Clara, CA) were digested with BglII and SalI. The fragments of interests were recovered by agarose gel analysis, purified and ligated using T4 DNA ligase to generate the expression plasmid pIRES2-EGFP-IDO. The recombinant expres- sion plasmid was confirmed by digestion with BglII and SalI and sequencing. CHO cells were cultured in RPMI medium 1640 with 10% FBS for 24 h and then trans- fected with 10 μg of pIRES2-EGFP-IDO using a stan- dard electroporation method (field strength of 350 V/ cm, 60 μs, 1 pulse). The pIRES2-EGFP vector was used as a plasmid control, and CHO cells transfected with pIRES2-EGFP (CHO/EGFP) were used as a control cell line. The CHO/EGFP cells were established as described previously [11]. G418 (1 mg/ml) was added to the med- ium 48 h after transfection, and the medium was chan- ged every 48 h for 4 weeks to obtain G418-resistant transformants. CHO cells containing pIRES2-EGFP-IDO were then identified by flow cytometric analysis. Detection of IDO gene transcripts in CHO cells and Foxp3 in co-cultured cells by RT-PCR To investigate IDO gene integration into CHO cells, total RNA was isolated from CHO cells transfected with pIRES2-EGFP-IDO using Trizol. RT-PCR primers were: IDO (188 bp), sense 5’-CATCTGCAAATCGTGAC- TAAG-3’; antisense 5’-CAGTCGACACAT- TAACCTTCCTTC-3’. b-actin (186 bp) was used as an internal control; sense 5’-TGGCACCCAGCACAAT- GAA-3’; antisense 5’-CTAAGTCATAGTCCGCCTA- GAAGCA-3’. cDNA was prepared by Oligo-(dT)15 from 1 μg of total RNA, and PCR was performed using a RT- PCR kit (Takara) according to the manufacturer’s instructions. To analyze Foxp3 gene expression in co- cultured cells, total RNA was isolated using Trizol as described above, with Foxp3 (488 bp) primers, forward primer 5’-CCCACTTACAGGCACTCCTC-3’; reverse primer 5’-CTTCTCCTTCTCCAGCACCA-3’. RT-PCR was performed in a volume of 20 μL using 50 ng of RNA, 2 μL of 10× PCR buffer (Takara, Japan), 10 mM of each deoxynucleoside triphosphate (dNTP), 1 μL of each primer, 0.5 μL of Takara Taq polymerase and 13.5 μL of water. Conditions were 94° for 5 min, followed by