The Role Of Lung Cancer Derived IDO1 In T Cell Exhaustion And Its Antitumor Immunotherapy

Background:The morbidity and mortality of lung cancer have ranked first among malignant tumors in the world.At present,the postoperative recurrence rate of lung cancer in Chinese population is high and the prognosis is poor.The 5-year survival rate of end-stage patients is less than 20%.Lung cancer has gradually become one of the most common and important diseases that seriously endanger the life and health of Chinese residents.Therefore,to find efficient and safe methods for the prevention and treatment of lung cancer has always been a major topic in cancer research.In recent years,it has been found that T cell exhaustion is accompanied by an abnormal phenomenon of T cell function in the process of tumor development.Recent studies have shown that T cell exhaustion occurs in melanoma-bearing mice,which is shown to be up-regulated by inhibitory receptor PD-1 and decreased secretion of cytokines IL-2 and TNF-?.Tumor occurrence and development is the process of mutual confrontation between tumor cells and immune cells,in particular,T cells plays a very important role,if the T-cell exhaustion will cause immune function abate,tumor immune tolerance will lead to invalidation of cancer immunotherapy,so further research on the characteristics of T cell exhaustion and its related mechanism will provide new ideas for tumor immunotherapy,not only has important theoretical significance,but also has significant clinical value.Indoleamine 2,3-dioxygenase is the important endogenous immune negative regulatory molecules in the body and IDO is intracellular heme,a kind of enzyme,is the only limit catalytic extrahepatic tryptophan along the way kynurenine catabolism.It has played a very important role in maintaining the body's immune tolerance,keeping steady-state in transplantation,forming tumor local immunosuppression microenvironment,and mediating tumor immune escape.Munn found that IDO1 was not only expressed in tumor-related cells,such as DC,macrophages and endothelial cells,but also highly expressed in tumor cells.Que Z found that IDO1 was expressed in non-small cell lung cancer of mice,and the increase of Treg cells was promoted by increasing the expression of IDO1,resulting in immunosuppression.In the mouse lung cancer experiment,Mittal and R found that the expressions of inhibitory receptors PD-1 and BTLA in CD4~+T cells and CD8~+T cells of lung cancer mice were higher than those of normal mice,and the cytokines IFN-?and IL-2 secreted by CD8~+T cells of lung cancer mice were lower than those of normal mice.Cara C.Schafer found in the study of mouse lung cancer model that IDO-/-knockout lung cancer mice could reduce the tumor size and reduce the expression of PD-1 depleted by tumor infiltrating CD4~+and CD8~+T cells,indicating that deficiency of IDO can inhibit T cell exhaustion.Previous studies have found that high expression of IDO1 in DC will increase expressions of PD-1?TIM-3?BTLA,causing T-cell exhaustion.Silencing the IDO1 in the DC will downgrade inhibitory receptors of T-cell and reduce the exhaustion of T cells.High expression of IDO1 in tumor cells can inhibit cytotoxic T cells,and promote the generation of Treg cells.Will the IDO1 in tumors and exhaustive T cells interact with each other?Whether the high expression of IDO1in tumors promotes T cell exhaustion is a question worthy of discussion.At present,the related studies on IDO1 and tumors mainly focus on the immune tolerance of tumors.However,there are few reports on the relationship between IDO1and exhaustive T cells in the tumor environment.IDO1 is highly expressed in lung cancer.In this study,lung cancer cell lines with low expression of IDO1 will be constructed to co-culture with exhaustive T cells to study the role of IDO1 expression and T cell exhaustion.In vivo,mice were inoculated with lung cancer cell lines with high expression of IDO1.Tumor-bearing mice model was established,and then the mice were treated with IDO1-sh RNA to observe the T cell exhaustion and tumor growth.In summary,we proposed the following hypotheses:(1)The high expression of IDO1 in lung cancer cells leads to the overexpression of T cell inhibitory receptors PD1 and BTLA,which leads to T cell exhaustion and promotes the growth of tumors.(2)The low expression of IDO1 in tumors in vivo can reverse T cell exhaustion and re-establish anti-tumor immunity,thus inhibiting the growth of lung cancer.This study was carried out from the following aspects:(1)The role of lung cancer-derived IDO1 in exhaustive T cells;(2)In vivo experimental study of low expression of IDO1 in lung cancer to inhibit T cell exhaustion and tumor growth,to observe the biological characteristics of oncogenic IDO1 on exhaustive T cells,so as to further explore the mechanism of action of IDO1 in lung cancer on T cell exhaustion,so as to provide a targeted basis for immunotherapy of lung cancer.Part 1 Study on the role of lung cancer-derived IDO1 in exhausted T cells in vitroObjective:To observe the effect of indoleamine 2,3-dioxygenase 1(IDO1)silencing on Lewis lung cancer cell line(LLC)by RNA interference(RNAi),and to verify the effect of down-regulation of IDO1 expression on T cell exhaustion.Methods:IDO1 si RNA was used to silence the expression of IDO1 in LLC cells.The expression of IDO1 m RNA was detected by real-time PCR,and the expression of IDO1 protein was detected by Western blot.The tumor bearing mice model of lung cancer LLC was established.T cells were isolated from lymph nodes of mice,and then compared with LLC cells treated with IDO1-si RNA and LLC cells treated with Gl2 si RNA.The proliferation and apoptosis of T cells were detected,and the expression of PD-1 and BTLA were detected.Results:The results of real-time PCR and Western blot showed that the m RNA and protein expression of IDO1 in LLC cells were significantly decreased after transfection with IDO1-si RNA;Compared with the negative control Gl2-si RNA group,the proliferation ability of T cells was significantly increased(Gl2-si RNA:74.4±1.5,IDO1-si RNA:82.5±2.3),and the apoptosis ability was significantly decreased(Gl2-si RNA:73.63±1.85,IDO1-si RNA:13.83±1.34).The expression of PD-1 and BTLA(PD-1:8.37±1.17%;BTLA:5.06±0.47%)in IDO1-si RNA treated LLC and T cells co cultured group was significantly lower than that in Gl2-si RNA treated LLC and untreated LLC cells co cultured group(PD-1:15.8±1.18%;BTLA:7.34±1.02%)and untreated LLC cells co cultured group(PD-1:16.8±2.3%;BTLA:7.83±1.07%).Conclusion:Silencing IDO1 expression in LLC cells in vitro can inhibit T cell apoptosis,promote T cell proliferation and also inhibit T cell exhaustion.Part 2 Inhibition of T cell exhaustion and tumor growth by low expression of IDO1 in lung cancer bearing mice in vivoObjective:To study the effect of low IDO1 knockdown on T cell exhaustion in lung cancer bearing mice and its growth.Methods: After establishing the model of LLC lung cancer bearing mice,the content of CD4+,CD8+ T cells in lymph nodes and spleen of lung cancer bearing mice and non tumor mice were detected by flow cytometry,and the expression of PD-1 and BTLA were detected by ELISA.The secretion of TNF-? and IL-2 were detected by ELISA,and then IDO1-sh RNA and Scramble-sh RNA plasmids were injected into lung cancer bearing mice through tail vein for therapy.The expression of IDO1 in lung cancer bearing mice was detected by immunohistochemistry.The tumor size was measured by vernier caliper and the weight of tumor was weighed by balance.At last,the inhibitory receptors PD-1 and BTLA were detected by flow cytometry,the secretions of TNF-? and IL-2 were detected by ELISA.Results:Compared with the normal mice,the expressions of PD-1 and BTLA in CD4+,CD8+ T cells were increased in lung cancer bearing mice;After systemic treatment with IDO-sh RNA,the growth rate of tumor in IDO1-sh RNA treatment group was significantly decreased,the size of tumor and the weight of tumor were also decreased significantly compared with that of Scramble-sh RNA and control group;The expression of silenced IDO1 inhibited the expressions of PD-1 and BTLA in CD4+,CD8+ T cells.In vivo IDO1-sh RNA treatment of tumor-bearing mice with lung cancer,the secretion of TNF-? and IL-2 increased,and could be restored to the secretion of TNF-? and IL-2 in normal mice(non-tumor).Conclusion:The growth of lung cancer in mice was inhibited by injecting IDO1-sh RNA into tail vein.The mechanism may be through the inhibition of T cell exhaustion.