title: Gastric cancer cells induce human CD4+Foxp3+ regulatory T cells through the production of TGF-β1.
abstract: OBJECTIVE
  To elucidate the molecular and cellular features responsible for the increase of regulatory T cells (Tregs) in gastric cancer.
  
  
  METHODS
  The frequencies of CD4(+)Foxp3(+) Tregs and the level of transforming growth factor-β1 (TGF-β1) were analyzed from 56 patients with gastric cancer by flow cytometry and enzyme-linked immunosorbent assay respectively. Foxp3 gene expression was analyzed by real-time polymerase chain reaction. The gastric cancer microenvironment was modeled by establishing the co-culture of gastric cancer cell line, MGC-803, with sorting CD4(+) T cells. The normal gastric mucosa cell line, GES-1, was used as the control. The production of TGF-β1 was detected in supernatant of MGC and GES-1. The carboxyfluorescein diacetatesuccinimidyl ester (CFSE) dilution assay was performed to evaluate the proliferation characteristics of induced Tregs. Neutralizing anti-TGF-β1 antibody was added to the co-culture system for neutralization experiments.
  
  
  RESULTS
  The level of serum TGF-β1 in gastric cancer patients (15.1 ± 5.5 ng/mL) was significantly higher than that of the gender- and age-matched healthy controls (10.3 ± 3.4 ng/mL) (P < 0.05). Furthermore, the higher TGF-β1 level correlated with the increased population of CD4(+)Foxp3(+) Tregs in advanced gastric cancer (r = 0.576, P < 0.05). A significant higher frequency of CD4(+)Foxp3(+) Tregs was observed in PBMCs cultured with the supernatant of MGC than GES-1 (10.6% ± 0.6% vs 8.7% ± 0.7%, P < 0.05). Moreover, using the purified CD4(+)CD25(-) T cells, we confirmed that the increased Tregs were mainly induced from the conversation of CD4(+)CD25(-) naive T cells, and induced Tregs were functional and able to suppress the proliferation of effector T cells. Finally, we demonstrated that gastric cancer cells induced the increased CD4(+)Foxp3(+) Tregs via producing TGF-β1. Gastric cancer cells upregulated the production of TGF-β1 and blockade of TGF-β1 partly abrogated Tregs phenotype.
  
  
  CONCLUSIONS
  Gastric cancer cell can induce Tregs development via producing TGF-β1, by which the existence of cross-talk between the tumor and immune cells might regulate anti-tumor immune responses.
authors.affiliation: Department of Laboratory Medicine, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China.
authors.forename: Xiang-Liang
authors.initials: XL
authors.lastname: Yuan
authors.forename: Lei
authors.initials: L
authors.lastname: Chen
authors.forename: Tong-Tong
authors.initials: TT
authors.lastname: Zhang
authors.forename: Yan-Hui
authors.initials: YH
authors.lastname: Ma
authors.forename: Yun-Lan
authors.initials: YL
authors.lastname: Zhou
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhao
authors.forename: Wei-Wei
authors.initials: WW
authors.lastname: Wang
authors.forename: Ping
authors.initials: P
authors.lastname: Dong
authors.forename: Liang
authors.initials: L
authors.lastname: Yu
authors.forename: Yan-Yun
authors.initials: YY
authors.lastname: Zhang
authors.forename: Li-Song
authors.initials: LS
authors.lastname: Shen
mesh_terms: D015704:CD4 Antigens / Q000276:immunology*; D045744:Cell Line, Tumor; D005260:Female; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000276:immunology*; D006801:Humans; D008213:Lymphocyte Activation / Q000276:immunology*; D008297:Male; D008875:Middle Aged; D013274:Stomach Neoplasms / Q000097:blood / Q000276:immunology*; D050378:T-Lymphocytes, Regulatory / Q000276:immunology*; D053773:Transforming Growth Factor beta1 / Q000097:blood / Q000276:immunology*
chemical_list: D015704:CD4 Antigens; C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D053773:Transforming Growth Factor beta1