title: Upregulated expression of indoleamine 2, 3-dioxygenase in CHO cells induces apoptosis of competent T cells and increases proportion of Treg cells.
abstract: BACKGROUND
  The inflammatory enzyme indoleamine 2, 3-dioxygenase (IDO) participates in immune tolerance and promotes immune escape of IDO+ tumors. A recent hypothesis suggested that IDO may contribute to the differentiation of new T regulatory cells (Tregs) from naive CD4+ T cells. In this study we investigated the role of IDO in induction of immunosuppression in breast cancer by increasing the apoptosis of T cells and the proportion of Tregs.
  
  
  METHODS
  An IDO expression plasmid was constructed and Chinese hamster ovary (CHO) cells were stably transfected with human IDO. Purified CD3+ T cells were isolated from the peripheral blood monouclear cells of breast cancer patients. After co-culturing IDO expressing or untransfected (control) CHO cells with T cells, T cells apoptosis were determined by flow cytometry analysis and annexin-V and PI staining. The proportion of the regulatory T cell (Tregs [CD4 + CD25 + CD127⁻]) subset was measured by flow cytometry analysis. T cells total RNA and cellular protein samples were isolated for detecting Foxp3 gene and protein expression.
  
  
  RESULTS
  IDO transgenic CHO cells yielded high levels of IDO enzymatic activity, resulting in complete depletion of tryptophan from the culture medium. We found that apoptosis occurred in 79.07 ± 8.13% of CD3+T cells after co-cultured with IDO+ CHO cells for 3 days and the proportion of CD4 + CD25 + CD127⁻ T cells increased from 3.43 ± 1.07% to 8.98 ± 1.88% (P < 0.05) as well. The specific inhibitor of IDO,1-MT efficiently reversed enhancement of T cells apoptosis and amplification of Tregs in vitro. Increased expression of Foxp3, a key molecular marker of Tregs, was confirmed by RT-PCR, real-time RT-PCR and Western blot analysis at the same time.
  
  
  CONCLUSIONS
  These results suggest that IDO helps to create a tolerogenic milieu in breast tumors by directly inducing T cell apoptosis and enhancing Treg-mediated immunosuppression.
authors.affiliation: Department of Breast Oncology, Tianjin Medical University Cancer Institute and Hospital, Tiyuanbei, Huanhuxi Road, Hexi District, Tianjin 300060, China.
authors.forename: Jingyan
authors.initials: J
authors.lastname: Sun
authors.forename: Jinpu
authors.initials: J
authors.lastname: Yu
authors.forename: Hui
authors.initials: H
authors.lastname: Li
authors.forename: Lili
authors.initials: L
authors.lastname: Yang
authors.forename: Feng
authors.initials: F
authors.lastname: Wei
authors.forename: Wenwen
authors.initials: W
authors.lastname: Yu
authors.forename: Juntian
authors.initials: J
authors.lastname: Liu
authors.forename: Xiubao
authors.initials: X
authors.lastname: Ren
mesh_terms: D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000201:enzymology / Q000276:immunology / Q000473:pathology*; D016466:CHO Cells; D049109:Cell Proliferation; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D005434:Flow Cytometry; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007108:Immune Tolerance; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000201:enzymology / Q000276:immunology / Q000473:pathology*; D050378:T-Lymphocytes, Regulatory / Q000201:enzymology / Q000276:immunology / Q000473:pathology*; D015854:Up-Regulation
chemical_list: C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase; D012333:RNA, Messenger