Study On The Expression Of IDO In MDSCs Derived From The Spleen Of Lewis Lung Carcinoma Bearing Mice

ObjectsTumor cells can induce distinct types of suppressor cells by several functionally distinct mechanisms. Among these suppressor cells, myeloid derived suppressor cells (MDSCs)have become a hotspot in the research of tumor-induced immune tolerance. MDSCs, phenotypically characterized in mice as CD11b+Gr-1+ cells, are comprised of immature macrophages, granulocytes, dendritic cells, and myeloid cells at earlier stages of differentiation. During tumor progression, a variety of cytokines such as GM-CSF, M-CSF, IL-3, IL-6, and VEGF are produced to prevent myeloid differentiation and result in expansion and accumulation of MDSCs, and it will be detrimental to anti-tumor immunity. Elucidation of the characteristics of MDSCs and their mechanisms of suppression will help develop new strategies of tumor immunotherapy.Indoleamine 2,3-dioxygenase(IDO)degrades the essential amino acid tryptophan in mammals, and broad evidence implicates that IDO and the tryptophan catabolic pathway contribute to the generation of tumor-induced immune tolerance. In particular, recent finds have established that IDO is overexpressed in both tumor cells and antigen-presenting cells in tumor-draining lymph nodes, where it promotes the establishment of immune tolerance to tumor antigens. Then whether IDO is expressed in MDSCs and whether MDSCs can suppress anti-tumor immunity via expressing IDO? To understand these questions, we sorted MDSCs from Lewis lung carcinoma bearing mice during tumor progression, detected the expresson of IDO in these MDSCs, and then investigated the affection of such MDSCs on T lymphocytes proliferation.Methods1. Fluorescence conjugated monoclonal antibodies and flow cytometry were used to detect the expression of surface markers CD11b and Gr-1 on the splenic mononuclear cells of Lewis lung carcinoma bearing mice at early and late tumor bearing stages or normal mice.2. Sorted the MDSCs from splenic mononuclear cells of Lewis lung carcinoma bearing mice at early and late tumor bearing stages or normal mice by utilizing immunomagnetic beads.3. RT-PCR was performed to detecte the expression of IDO mRNA and Western Blot was performed to detect the expression of IDO protein in MDSCs from the spleen of Lewis lung carcinoma bearing mice at early and late tumor bearing stages or normal mice.4. Supernatants of MDSCs from Lewis lung carcinoma bearing mice at early and late tumor bearing stages were harvested and were analysed for kynurenine concentration by HPLC.5. To detect the influence of MDSCs from Lewis lung carcinoma bearing mice at early and late tumor bearing stages on T cell proliferation by WST-1 assay.Results1. The percentage of CD11b/Gr-1 double positive cells in the splenic mononuclear cells of Lewis lung carcinoma bearing mice at early and late tumor bearing stages or normal mice were (20.00±5.28)%, (33.90±2.74)%, (4.07±2.42)%, respectively.2. IDO mRNA and protein were expressed in MDSCs from both Lewis lung carcinoma bearing mice and normal mice, while only few IDO mRNA and protein were expressed in MDSCs from the spleen of Lewis lung carcinoma bearing mice at early tumor bearing stages and normal mice, and the expression of IDO mRNA and protein increased in MDSCs from the spleen of Lewis lung carcinoma bearing mice at late tumor bearing stages.3. Few kynurenine was detected in supernatants of MDSCs from the spleen of Lewis lung carcinoma bearing mice at early tumor bearing stages(5.882±1.863), however, kynurenine levels increased in supernatants of MDSCs from the spleen of Lewis lung carcinoma bearing mice at late tumor bearing stages(22.746±6.997), and the difference between two groups was significant (P<0.05).4. MDSCs from the spleen of Lewis lung carcinoma bearing mice at late tumor bearing stages could significantly suppress T cell proliferation compared with MDSCs from the spleen of Lewis lung carcinoma bearing mice at early tumor bearing stages, the inhibition ratios were (44.51±5.55)% vs. (11.08±4.86)%, P<0.01. This suppressive effect of MDSCs from the spleen of Lewis lung carcinoma bearing mice at late tumor bearing stages on T cell proliferation was prevented by adding the IDO inhibitor 1-MT, the inhibition ratios were (20.00±1.68)% vs. (44.51±5.55)%, P<0.01.However, the presence of 1-MT had no effect on T cell proliferation, as for MDSCs from the spleen of Lewis lung carcinoma bearing mice at early tumor bearing stages, and there was no statistically significant difference between two groups(P>0.05).Conclusions1. IDO was expressed in MDSCs from the spleen of Lewis lung carcinoma bearing mice, and the expression of IDO was significant higher in MDSCs from the spleen of Lewis lung carcinoma bearing mice at late tumor bearing stages.2. MDSCs from the spleen of Lewis lung carcinoma bearing mice at late tumor bearing stages were capable of exerting a T cell suppressive effect, which might be mediated by IDO through the depletion of tryptophan.