Upregulated expression of indoleamine 2, 3-dioxygenase in CHO cells induces apoptosis of competent T cells and increases proportion of Treg cells

Abstract

Introduction. 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. © 2011 Sun et al; licensee BioMed Central Ltd.