Research On The Mechanism Of Indoleamine 2, 3-Dioxygenase In Tumor-induced Tolerance Through CD4~+CD25~+CD127~-/Foxp3~+ T Cells Generation

Objects: This study aimed to evaluate the potential correlation between indoleamine 2,3-dioxygenase and local infiltrative regulatory T cells in primary female breast cancer patients. Establishing a IDO transgenic CHO cell line and investigating the role of IDO in the development and apoptosis process of Tregs. Methods: From June 2007 to August 2007, 20 primary female breast cancer patients received modified mastectomy and had complete data. The expression of IDO from the tissues of breast neoplasms, normal breast tissue and the tumor-draining lymph nodes (TDLNs) was identified by RT-PCR analysis. And Live Cells were isolated from the tissues mentioned above. The proportion of CD₄⁺CD₂₅⁺CD₁₂₇^- T cells population was evaluated by flow-cytometry analysis. To investigate the contribution of high expression of IDO to the local infiltrative Tregs. The expression level of CD4, IDO and Foxp3 were examined by immunohistochemistry for further evaluation of the correlation between IDO and Tregs in breast cancer patients. A 1225kb fragment was encompassing the entire coding region of human INDO cDNA was obtained using RT-PCR kit. The PCR products were inserted into pMD19-T Simple Vector by TA-cloning procedures, resulting in pMD19-IDO followed by sequencing identification. The genes were then cut from pMD19-T vectors and cloned into eukaryotic expression plasmid pIRES2-EGFP. CHO cells were then transfected with the recombinant pIRES2-EGFP-IDO plasmid using standard electroporation method. Stable transformants were obtained by G418 selection. The integrated INDO genes were determined by PCR analysis. And the transcription and expression of INDO gene were detected by RT-PCR and Western blot analysis, respectively. The enzyme activity of IDO were measured using Hitachi amino acid L-8800-automatic analyzer (Hitachi, Tokyo, Japan). Mononuclear cells was isolated from the peripheral blood of breast cancer patients using CS-3000 Plus Blood Cell Separator (Baxter, Munich, Germany). CD3⁺T cells were isolated and purified using RosetteSep Human CD3 Depletion Cocktail kit. Then Coculture of CD3⁺ T cells with INDO gene-integrated CHO cell, pIRES2-EGFP transfected CHO cells as blank plasmid control. The apoptosis of the T cells was analyzed by Annexin V-FITC and PI staining method. And the Tregs subsets were defined as CD4⁺CD25⁺CD127^- examined by flow cytometry analysis. The mRNA expression of Foxp3 was analyzed by semi quantitative and real time quantitative RT-PCR analysis. And the expression levels of Foxp3 were detected by Western blotting. Results: IDO expression in TDLNs was higher than that in primary breast neoplasms, while IDO expression in normal breast tissue was the lowest (P<0.05). More infiltrative CD₄^-CD₂₅⁺CD₁₂₇^- T cells were found in TDLNs. The proportions of Tregs population detected in TDLNs, breast neoplasms and normal breast tissue were (4.40±2.91)%,(2.62±2.14)%,(2.01±1.49)%, respectively (P<0.05). Linear correlation between IDO and Foxp3 was observed according to immunohistochemical stain (P<0.05). The recombinant expression plasmid pIRES2-EGFP-IDO was confirmed by Bgl II and Sal I digestion. The expression of IDO in transfected CHO cells was identified by RT-PCR and Western blotting. And the IDO transgenic CHO cells yielded a high enzyme activity and resulted in complete depletion of tryptophan from the culture medium. 72 h after co-culture of CD3⁺T cells and CHO cells with IDO transfection, the apoptotic rate of CD3⁺T cells was (79.07±8.13)%, while that in CHO cells with mock plasmid transfection was (59.80±11.46)%. The apoptotic rate of CD3⁺T without co-culture was (32.40±6.40)%. The differences were statistically significant (P<0.05). 7 days after co-culture, the percentage of CD₄⁺CD₂₅⁺CD₁₂₇^- T cells in CD₄⁺ T cells was (8.98±1.88)%, but the Tregs was only (3.73±1.12)% in CD3⁺T cells co-cultured with CHO cells with mock plasmid tranfection (P<0.05). Foxp3 expression increased after co-culture with INDO gene-integrated CHO cells according to the semi quantitative RT-PCR analysis, so that was confirmed by real time quantitative RT-PCR analysis (P<0.05). 7 days after co-culture, the ratio of Foxp3 protein expression/β-actin was (0.0450±0.0025) in CD3⁺T cells incubation with INDO gene-integrated CHO cells, while Foxp3 protein could not be detected in CD3⁺T cells incubation with CHO cells with mock plasmid transfection and in CD3⁺T without co-culture. Conclusion: IDO expression in the tissues is accompanied with the infiltration of Tregs, indicating a close relation between IDO activity and Tregs in breast cancer patients. The genes encoding INDO have been successfully expressed in eukaryotic expression system. And the CHO cell lines with stable IDO expression have been established successfully. The enzymatic activity of IDO results in the reduction in local tryptophan concentration and the production of tryptophan metabolites, such as kynurenine. IDO can indeed affect the proliferation process of T cell and induce T cell apoptosis in vitro, indicating IDO mediated immune tolerance should comprise the process of IDO-induced T cell apoptosis. IDO can induce the maturation and differentiation of Tregs cell in vitro. IDO may facilitate the differentiation of cells with differentiation capacity among CD3⁺T cells into CD4⁺CD25⁺CD127^- cell. IDO as an endogenous mechanism is involved in a variety of peripheral tolerance and immunosuppression response, its role requires joint participation of other mechanisms, such as the regulation of IDO on Tregs cell. In a word, our study investigates the relationship and the interaction between IDO and Tregs cells. And the results indicate a close relation between IDO activity and the infiltration of Tregs in breast cancer patients. And the further experiments suggest that there are several mechanisms underlying IDO-induced tolerance process, including the exhaust of tryptophan and the toxic effect of its metabolite, leading to the apoptosis of T cells, and the potential effects of IDO on the genesis and development of Tregs cells.