The Proliferation Of Regulatory T Cell Induced By Mouse Lewis Lung Carcinoma Cell Transfected With IDO Gene

Background and objective:Metastasis is one of the poor prognostic factors of non-small cell lung cancer(NSCLC). One of the most important causes is immune tolerance within the tumor microenvironment induced by many factors such as regulatory T cell populations,inhibitory ligands such as PD-L1,soluble factors such as TGFb,and the activity of nutrient-catabolizing enzymes such as indoleamine 2,3-dioxygenase (IDO). Increasing evidences are accumulating to indicate that IDO and regulatory T cell(Treg) play an important role in the induction of immune tolerance in tumor microenvironment.However the relationship between the expression of IDO and the distribution of Tregs in NSCLC has not been completely elucidated. The present study aimed to investigate the IDO expression in NSCLC and the distribution of Tregs in NSCLC tissues and the metastastic lymph nodes,and the potential relationships between them.Besides that,we investigate the effects of mouse Lewis lung cancer cell line transfected with IDO gene on the induction of the proliferation of Treg cell in vitro and in vivo and the effects of IDO(+) lung cancer cell on the invasion and metastasis of mouse Lewis lung cancer cell,thereby exploring its possible mechanisms.1-Methyl tryptophan(1-MT), the IDO competitive inhibitor, was tried to reverse the proliferation of Treg cell.Methods:Sixty two patients with NSCLC and fifteen patients with benign lung disease from Xinqiao hospital and Southwest hospital were involved in the present study. The IDO expression in lung cancer, the peritumorous normal lung tissues and the benign control tissues was detected by streptavidin-peroxidase(SP) immunohistochemistry (IHC) and fluorescent semi-quantitative RT-PCR. The distribution of Treg cell was detected by SP IHC as well. With the use of the cationic liposome Lipofectamine 2000,the eukaryotic expression plasmid vectors pEGFP-N1 carrying human IDO cDNA was transfected into mouse Lewis lung cancer cell line(named Lewis-IDO) and then co-cultured with T lymphocytes from the peripheral blood of C57 mouse.The parental Lewis cells and Lewis cells transfected with blank plasmid pEGFP-N1(named Lewis-EGFP) was used as control groups.After co-culture,the Treg cells were sorted using fluorescence-activated cell sorting (FACS). The capability of invasion and metastasis in vitro was studied by using of transwell experiment. To evaluate the tumorgenicity and metastastic ability of IDO gene on cancer cell,the Lewis-IDO, Lewis-EGFP and Lewis cells were transplanted in C57 mouse respectively. The Tregs in the peripheral blood(PB) and cancer mass were detected by FACS. 1-MT was used in the drinking water of mouse with a concentration of 5mg/ml.Results:1. The positive IDO expression rates in lung cancer cell, corresponding normal lung tissues and benign disease tissues were 67.7% (42/62),0 and 13.3% (2/15) , respectively.There were significant differences between cancer mass and benign disease tissues, peritumorous normal lung tissues(P<0.01).The positive IDO expression rate in metastastic lymph node was 75.0%(15/20). The results of fluorescent semi-quantitative RT-PCR were consistent with the results of IHC. The percentage of Treg cells in lung cancer tissues, metastastic lymph node, peritumorous normal lung tissues and benign disease tissues were 83.9%(52/62),90.0%(18/20),0 and 13.3%(2/15), respectively. Significant differences were also seen between the former two and the latter two(P <0.01),but there was no significant differences between the two formers or the two latters(P >0.05). The spearman rank correlation analysis revealed the distribution of Treg cells is correlated with the expression of IDO(R=0.448,P <0.01).2. The IDO gene was transfected into Lewis cell successfully. Compared with the Lewis-EGFP and Lewis cells,Lewis-IDO cells became longer and had more filopodia.By transwell chamber system,the invasion capability of Lewis-IDO cells was significantly increased. The quantity of the Lewis-IDO cells breaking through the matrigel increased significantly(P<0.05). The Lewis-IDO cells could induce the proliferation of Treg cells.There was significant differences between the Lewis-IDO cells and Lewis-EGFP cells(6.2±0.36% vs 5.0±0.52%,P<0.05) and between the Lewis-IDO cells and Lewis cells(6.2±1.6% vs 4.9±0.4%,P<0.05).3. The time of tumor formation was different among the three groups but there were no significant differences.The capability of metastasis to lung and liver of Lewis-IDO cell was significantly higher than that of Lewis-EGFP cells or Lewis cells(P =0.01) The Treg cells in PB of the mouse transplanted with Lewis-IDO cells were statistically higher than that from the mouse transplanted with Lewis-EGFP cells or Lewis cells(P <0.05),the values are (13.7±4.5)%, (8.9±3.3)% and (9.2±3.4)% respectively.The mean survival time of Lewis-IDO(37.1±2.3 days) group was significantly longer than Lewis-EGFP group(45.1±2.6 days) or Lewis group(44.9±2.9 days) (P <0.05).4. After the treatment with 1-MT, the IDO expression was inhibited by IHC.The proliferation of Treg cell was significantly reduced than untreated group( (9.7±4.0) % vs (13.7±4.5)%,P <0.05). The metastasis to lung and liver was significantly reduced than untreated Lewis-IDO group(P <0.05).The mean survival time(42.0±2.3 days) of C57 mouse was longer than that of Lewis-IDO group(37.1±2.3 days) but there was no significant difference(P >0.05).Conclusions:1. The overexpression of IDO is a common molecular abnormality both in lung cancer and in metastasic lymph node and is associated with the proliferation of Treg cell in tumor microenvironment of NSCLC.It implicates that the IDO(+) cancer cell might be concerned with the tumor immune tolerance by inducing the proliferation of Treg cells in tumor microenvironment and suggests that strategies to regulate the IDO activity may be beneficial in the immunotherapy of human NSCLC.2. The tranfection of IDO gene into Lewis lung cancinoma cell can induce the proliferation of Treg cells and increase the Lewis cell's capability of invasion and metastasis both in vitro and in vivo which implicate that IDO is a new treatment target for reverse the immune tolerance of NSCLC and inhibit the invasion and metastasis of NSCLC.3. 1-MT treatment can significantly reduce the metastasis of NSCLC and the proliferation of Treg, but can not completely inhibits the IDO expression and significantly prolong the mean survival time of mouse.It is necessary to investigate more effective and specific methods to inbibit IDO-mediated immune tolerance..