Study On The Role And Mechanism Of Antigen Presentation Of Exosomes Derived From Lung Epithelial Cells Irradiated By ⁶⁰Co γ Ray

Objective Radiation-induced lung injury(RILI)is a common complication of radiation therapy and nuclear radiation accidents,generally manifested as early acute radiation pneumonia and late radiation-induced pulmonary fibrosis.The previous research results of our laboratory showed that T lymphocytes(T cells)in lung tissue showed obvious phase change in the early stage after chest irradiation in mice,and confirmed that radiation damaged cells activated T cells through the antigen-presenting function of dendritic cells(DCs).In the further study of the antigen-presenting mechanism of radiation-injured cells,we observed that in the absence of DCs,the irradiated lung epithelial cells still activated the na?ve T cells.It is suggested that there may be a direct antigen-presenting pathway independent of professional antigen-presenting cells in radiation-injured lung epithelial cells.Based on literature research,we speculate that exosomes(exo)may be the key to direct antigen presentation of radiation-injured cells.Therefore,the purpose of this study is to investigate the activation effect and mechanism of T lymphocytes by irradiated mouse alveolar epithelial cells(MLE-12)and their exosomes(exo/IR-MLE).It is expected to provide experimental basis for a new way of clinical treatment of RILI.MethodsⅠ.Na?ve T cells isolated from C57BL/6 mouse spleen by immunomagnetic beads.Its purity and activity were identified by Flow cytometry(FCM).Tn were used for coculturing with MLE-12 cells(irradiated or not)treated(or not)with the exosome inhibitor GW4869 for 12 and 24 h.Then,FCM and cell counter were used to detected T cell subsets(CD3,CD4,and CD8)and their activation indicators(CD28 and CD69).Ⅱ.Exosomes were extracted by ultracentrifugation from the supernatants of a MLE-12 cell culture at 48 h after culturing.Western blot(WB),nanoparticle tracking analysis(NTA)and transmission electron microscope(TEM)were used for qualitative and quantitative analysis of exosomes.FCM and WB were used to identify the characteristic expression molecules(CD63,TSG101,CD81,and Calnexin,)and antigen presentation related molecules(MHC I,MHC II,CD80,and CD86)in exosomes.The exosomes(exo/IR-MLE and exo/NC-MLE)from MLE-12 cells(irradiated/non-irradiated)were cocultured with Tn for 12 and 24 h.T cell subsets(CD3,CD4,and CD8)and their activation indicators(CD28 and CD69)were detected by FCM and cell counter.Ⅲ.T cells treated with exo/IR-MLE coculturing with MLE-12 cells for 24 h.FCM was used to detect the change of apoptosis ratio of MLE-12 cells.ResultsⅠ.The results of co-culture of MLE-12 cells and Tn showed that,compared with the control group,T cells in the irradiation group proliferated;the proportion of CD4+cells in total T cells first decreased and then increased,the proportion of CD8+ cells first increased and then decreased,and the ratio of CD4/CD8 first decreased and then increased;costimulatory molecules CD28 and early activation index CD69 were highly expressed;GW4869 inhibited these activation and proliferation phenomenon.Ⅱ.After gamma ray irradiation,the number of exo/IR-MLE produced from a single MLE-12 cell clearly increased following a good dose effect relationship.TEM and NTA showed that exo/IR-MLE had a typical saucer like structure and particle size of30～150 nm.They showed high expression of exosome marker proteins such as CD63,CD81,and TSG101.Compared with control group,increased antigen presenting related molecules(MHC I,MHC II,CD80,and CD86)and some exosome markers’ expression(TSG101 and CD81)was detected by FCM and WB in exo/IR-MLE.Ⅲ.The results of co-culture of exosomes and Tn showed that,compared with the control group,T cells in the irradiation group proliferated;the proportion of CD4+cells in total T cells decreased,the proportion of CD8+ cells increased,and the ratio of CD4/CD8 decreased;costimulatory molecules CD28 and early activation index CD69 were highly expressed.Ⅳ.Compared with the control group,T cells treated with exo/IR-MLE could increase the apoptotic proportion of normal MLE-12 cells.ConclusionsIn this study,we simulated the immune microenvironment of lung tissue after thoracic irradiation in mice by cell coculture and found that irradiated MLE-12 cells could directly activate T cells via exosomes.T cells activated through this pathway have significant cytotoxic effects on normal MLE-12 cells.The expression of antigen presenting related molecules(MHC I/II,CD80/86,etc.)in the exosomes derived from MLE-12 cells damaged by radiation increases.This study preliminarily explored the mechanisms of T cell activation by alveolar epithelial-derived exosomes,providing new experimental evidence for the occurrence and development of radiation lung injury.