Abstract
IntroductionThe 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.MethodsAn 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.ResultsIDO 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.ConclusionsThese results suggest that IDO helps to create a tolerogenic milieu in breast tumors by directly inducing T cell apoptosis and enhancing Treg-mediated immunosuppression.
Highlights
The inflammatory enzyme indoleamine 2, 3-dioxygenase (IDO) participates in immune tolerance and promotes immune escape of IDO+ tumors
We investigated the potential effects of IDO on development of T regulatory cells (Tregs) cells in breast cancer tumors using a stable IDO-expressing Chinese hamster ovary (CHO) cell line
Analysis of IDO expression by PCR using genomic DNA, or by RT-PCR using total RNA, yielded a 188 bp fragment; no IDO expression was detected in CHO/EGFP cells, indicating that we could detect the integration into the CHO cell genome and transcription of the transfected IDO gene (Figure 1B)
Summary
The inflammatory enzyme indoleamine 2, 3-dioxygenase (IDO) participates in immune tolerance and promotes immune escape of IDO+ tumors. 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. Reduction in the local tryptophan concentration and generation of tryptophan metabolites 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]. 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. The role of increased IDO expression in tumor cells in development of Treg cells is not clear. 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
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