The Role Of Autoantibodies In Lung Injury Induced By Radiation Combined With Immune Checkpoint Inhibitors

Objective: In recent years,tumor immunotherapy has developed rapidly,among which immune checkpoint inhibitors have become a hot spot in the field of tumor therapy,and have achieved very good results in the treatment of a variety of tumors.However,with the comprehensive clinical application of ICIS,the immune related adverse events have gradually attracted people’s attention.These immune related adverse events mainly include skin,lung,endocrine,blood,digestive system,etc.among them,checkpoint inhibitor pneumonia(CIP)is a kind of ICI related toxicity.Immune related adverse reactions have unique characteristics,including organ specific propensity,no dosedependent correlation,and potential late onset.In order to explore the mechanism of lung injury induced by radiation therapy combined with anti-pd1,we used the model of lung injury induced by radiation plus PD-1 immunotherapy in mice,hoping to have new inspiration for clinical treatment.Methods: Forty c57bll6 male mice aged 6-8 weeks were randomly divided into four groups:(1)IgG control group(IgG group);(2)anti-PD-1 antibody group(apd-1group);(3)lung irradiation group(LR + IgG group);(4)anti-PD-1 antibody combined with lung irradiation group(apd-1 + LR group).The pathological changes of lung tissue were observed by HE staining.Immunoblotting method was used to verify whether the serum of mice in each group contained autoantibodies that could bind to mouse lung protein.Immunofluorescence and other methods were used to observe which autoantibodies of mice with lung injury combined with lung cell protein.The changes of TGF-β,TNF-α,IL-6,IL-17,STAT3,STAT6 and other cytokines and transcription factors were studied by ELISA,RT-PCR and flow cytometry.At the same time,by clearing the B cells in mice,blocking the production of auto IgG antibody,to determine whether it can improve the lung injury.Results: 1.Two weeks later,mild congestion and bleeding appeared in the lung tissue of the mice in the apd-1 + LR group,a small amount of red blood cell exudation in the lung interstitium,destruction of alveolar wall and vascular endothelium,and a small amount of inflammatory cells infiltration in the alveolar cavity and bronchioles.Four weeks later,in the apd-1 + LR group,the alveolar septum was broken,a large number of alveoli were fused,and some alveoli were collapsed;the alveolar wall was thickened,and a large number of inflammatory cells were infiltrated.The other three groups had no similar lung injury.2.The results of immune blot showed that at 2 weeks,the bands of apd-1 + LR group were deeper than those of the other three groups,and there was autoantibody IgG in the serum of apd-1 + LR group mice,and the IgG antibody could specifically bind to lung protein.At 4 weeks,the bands of apd-1 + LR group were deeper than those of the other three groups.3.Immunofluorescence results showed that there were a lot of IgG fluorescence deposition around the damaged alveolar epithelial cells in the apd-1 + LR group at 2weeks,and the IgG fluorescence deposition was more obvious at 4 weeks.The other three groups did not see similar IgG fluorescence deposition.However,Ig M did not deposit in all groups at 2 and 4 weeks.4.The results of ELISA and RT-PCR showed that at 2 weeks,the levels of TGF-β and TNF-α in apd-1 + LR group were significantly higher than those in the other three groups,and the difference was statistically significant,but there was no significant difference between apd-1 group and IgG control group,or between LR + IgG group and IgG group;at 4 weeks,the levels of TGF-β and TNF-α in apd-1 + LR group were higher than those in the other three groups,and the difference was statistically significant.The levels of TGF-β and TNF-α in LR + IgG group were higher than those in IgG group,and the difference was statistically significant.5.RT-PCR results showed that the transcription factors of CD19,CD20,T-bet and STAT3 in apd-1 + LR group were significantly higher than those in the other three groups at 2 and 4 weeks(P < 0.05),but there was no significant difference between apd-1 group and IgG group,or between LR + IgG group and IgG group.The transcription factor STAT-6 of apd-1 + LR group was significantly higher than that of the other three groups at 2 and 4 weeks(P < 0.05).Compared with IgG group,the level of STAT-6 in apd-1 group was significantly higher(P < 0.05).The levels of IL-6 and IL-17 in the apd-1 + LR group were also significantly higher than those in the other three groups(P < 0.05).Meanwhile,the levels of IL-6 and IL-17 in the apd-1 + LR group were also significantly higher than those in the IgG group and LR + IgG group(P < 0.05).6.After injection of anti-CD20 antibody,B cells in liver,lymph node,spleen and peripheral blood of mice can maintain a low state,and the decline rate in various organs is different,and all changes with time.7.Blocking the production of auto IgG antibody after clearing B cells can further significantly improve the lung injury caused by radiation combined with anti-pd1 antibody.Conclusion:Single dose of 6 Gy lung irradiation or anti-PD-1 treatment alone did not cause lung injury.But radiation combined with anti-pd1 antibody can cause obvious lung injury.IgG antibody that can specifically bind to lung protein can be detected in the serum of mice.Immunofluorescence showed that there was a large amount of IgG deposition in the lung tissue of mice.Further study showed that IL-6 was significantly increased in apd-1 + LR group,and IL-6 could transmit B cell proliferation signal through Stat3.At the same time,STAT6 and TGF-β also increased significantly,which played an important role in further promoting pulmonary fibrosis.However,blocking the production of autoing antibody after clearing B cells can further significantly improve the lung injury caused by radiation combined with anti-pd1 antibody.