BACKGROUND AND PURPOSE:Radiotherapy combined with immunotherapy has been shown to improve thoracic tumor outcomes while increasing the risk of lung injury. Low-dose radiotherapy (LD-RT) has been proven efficient in managing inflammatory diseases. This study aims to investigate whether LD-RT might alleviate lung injury induced by radiotherapy combined with immunotherapy, and attempt to explore its underlying mechanisms thereby offering novel insights for clinical application. METHODS:To establish a mouse model of lung injury caused by radiotherapy combined with immunotherapy, C57 BL/6J mice received intraperitoneal injections of programmed death 1(PD-1) inhibitor weekly and a single dose of 15 Gy whole thoracic irradiation. Then they received a single dose of LD-RT at 1.0 Gy or 1.5 Gy on Day 14 or 28. The mice were euthanized on Day 42, and lung tissue samples were collected for HE, Masson's trichrome, and immunohistochemical staining to evaluate lung tissue damage, fibrosis, and lymphocyte infiltration. The expression levels of cytokine were quantified by the enzyme-linked immunosorbent assay. RESULTS:Both low-dose of 1.0 Gy and 1.5 Gy attenuated lung injury caused by radiotherapy combined with PD-1 inhibitors, but 1.5 Gy was more effective. Compared with Day 14, LD-RT at 1.5 Gy on Day 28 was more effective in alleviating alveolar inflammation and reducing collagen deposition, and inhibiting lymphocyte infiltration and secretion of inflammatory cytokine in lung tissue. CONCLUSION:Low-dose radiation alleviated lung injury caused by radiotherapy combined with PD-1 inhibitor, and the alleviating effect is closely related to the timing and dose of the radiotherapy administered.

Abstract Background PD-1 inhibitors may superimpose the toxicity of radiotherapy while increasing the antitumor effect. However, there are fewer studies on immune myocarditis caused by radiotherapy plus anti-PD-1 and the mechanism is still under exploration. Methods 40 C57BL/6 mice were randomly assigned to 4 groups. A: Control, B: PD-1 inhibitor, C: cardiac irradiation and D: thoracic irradiation + PD-1 inhibitor, mice were treated with either anti-PD-1 antibody with or without thoracic radiation (15Gy). Each group contained ten mice, five of which were studied for a duration of 1 month and the remaining five for 3 months. Tunel staining was utilized to observe apoptosis of cardiac tissues; histological analysis was performed to analyze the structural and morphological alterations, fibrosis of the heart tissue. The infiltration of CD3+, CD4+, and CD8 + T-cells into the cardiac was analyzed through flow cytometry; Elisa measured the expression levels of TNF-α, IL-1β, and TLR-4 in the cardiac; and immunoprotein blotting and qPCR were used to observe the protein and mRNA expression levels of HMGB1, TLR-4, and NF-κB p65. Results Group D exhibited a greater degree of cardiac injury, fibrosis, and apoptosis in comparison to groups A, B, and C. Additionally, there was an increase in injury, AI, and CVF values after three months as opposed to one month (P < 0.05).After one month, there was no statistically significant difference in cardiac damage, AI, or CVF values between groups A and B; however, after three months, there was a significant difference (P < 0.05). Group D also had higher levels of IL-1β, IL-6, TNF-α and T-lymphocyte distribution, HMGB1, TLR4, NF-κB P65 protein, and mRNA expression than the other three groups. However, each group's index expression declined over the course of three months as opposed to one month, and this difference was statistically significant (P < 0.05). Conclusion PD-1 inhibitors exacerbated myocardial injury based on radiation by upregulating the expression of inflammatory factors in the HMGB1 signaling pathway. In the early stages of myocardial damage, inflammatory alterations predominated, while in the later stages, fibrosis.

e14631 Background: Radiation in combination with programmed cell death 1 (PD-1) antibodies greatly enhanced the anti-tumor activity by reactivating T lymphocytes. Whether it will produce the superposition of toxic side effects and the underlying mechanism remains elusive. Pyroptosis is a novel type of programmed cell death associated with the pathogenesis of many inflammatory diseases. We hypothesized that GSDMD plays a pathogenic role in PD-1 inhibitor combined with radiation-induced myocardial injury (RIMI) and that GSDMD gene knockout (KO) or blockade the caspase-1/GSDMD pathway will alleviate RIMI. Methods: GSDMD-KO mice and their wildtype (WT) C57BL/6J were used to investigate whether PD-1 inhibitor aggravated RIMI through pyroptosis pathway. Myocardial contractile functions, myocardial inflammation and fibrosis, and myocardial injury were assessed. Pyroptosis-related proteins and inflammatory cytokines were evaluated. Flow cytometry was used to detect the level of lymphocyte in myocardium. To further confirm the presence of pyroptosis in cardiomyocyte, double-immunostaining was performed. We verified the effect of radiotherapy combined with PD-1 inhibitor on pyroptosis in cell experiments. Results: Compared with irradiation or anti-PD-1 alone, PD-1 inhibitor could aggravate RIMI, increase the infiltration of CD8+T cells in the myocardium, and aggravate inflammatory reactions. The expressions of pyroptosis-related proteins and inflammatory cytokines were upregulated. Immunofluorescence also indicated that pyroptosis cells were increasing. While GSDMD-KO or caspase-1GSDMD inhibitor, myocardial injury and pyroptosis-related proteins were significantly reduced. Conclusions: We speculate that PD-1 inhibitors activate CD8+T cells and mediate cardiomyocyte pyroptosis. GSDMD-triggered immune-inflammatory response plays a key role in the aggravation of myocardial injury.

Purpose Programmed cell death protein 1 (PD-1) inhibitors may further increase the risk of cardiotoxicity of radiotherapy while improving the outcomes of locally advanced lung cancer. However, few studies have focused on cardiac injury caused by radiotherapy plus anti-PD-1 therapy, and the underlying mechanism is still under exploration. This study aimed to explore this mechanism. Methods Six- to eight-week-old C57BL/6 mice were treated with either an anti-PD-1 antibody or phosphate-buffered saline (PBS) with or without 15 Gray (Gy) cardiac irradiation (IR). Five mice were sacrificed at 1 month, and the remaining mice were sacrificed at 3 months. Histological analysis was performed to determine the structural and morphological alterations and cardiac fibrosis. The infiltration of cardiac T cells was analysed via flow cytometry, and western blotting and qPCR were used to detect the protein and mRNA expression levels of HMGB1-related pathway. Results Group D (IR + anti-PD-1) demonstrated more severe injury, fibrosis, and apoptosis compared to groups A (control), B (anti-PD-1), and C (IR). Furthermore, the injury observed in Group D was significantly more severe, with higher values of apoptotic index (AI) and fibrotic area at 3 months compared to 1 month (P < 0.05). At 1 month, there were no significant differences in cardiac damage or AI or CVF values between groups A and B, but these differences emerged at 3 months (P < 0.05). Group D exhibited greater infiltration of T lymphocytes and increased expression of high mobility group box-1 protein (HMGB1), Toll-like receptor 4 (TLR4), and nuclear factor kappa-B (NF-κB P65) at both 1 and 3 months compared to the other three groups. Conclusion In combination with radiation, PD-1 inhibitors exacerbated myocardial injury by modulating the HMGB1/NF-κB signalling pathway.

Cancer therapy has entered a new era with the use of programmed cell death protein 1 (PD-1) immune checkpoint inhibitors. When combined with thoracic radiotherapy, it demonstrates synergistic anti-tumor effects and potentially worsens radiation-induced myocardial fibrosis (RIMF). RIMF is the final stage of radiation-induced heart disease (RIHD) and a potentially fatal clinical complication of chest radiotherapy. It is characterized by decreased ventricular elasticity and distensibility, which can result in decreased ejection fraction, heart failure, and even sudden cardiac death. Pyroptosis, a type of programmed cell death, is mediated by members of the gasdermin (GSDM) family and has been associated with numerous cardiac disorders. The effect of pyroptosis on myocardial fibrosis caused by a combination of radiotherapy and PD-1 inhibitors remains uncertain. In this study, a 6MV X-ray of 20 Gy for local heart irradiation was used in the RIHD mouse model. We noticed that PD-1 inhibitors aggravated radiation-induced cardiac dysfunction and RIMF, concurrently enhancing the presence of CD8+ T lymphocytes in the cardiac tissue. Additionally, our findings indicated that the combination of PD-1 inhibitor and thoracic radiation can stimulate caspase-1 to cleave GSDMD, thereby regulating pyroptosis and liberating interleukin-8 (IL-18). In the myocardium of mice, the manifestation of pyroptosis mediated by GSDMD is accompanied by the buildup of proteins associated with fibrosis, such as collagen I, transforming growth factor β1 (TGF-β1), interleukin-6 (IL-6), vascular endothelial growth factor (VEGF), and tumor necrosis factor α (TNF-α). Moreover, it was discovered that TFG-β1 induced the phosphorylation of Smad2/Smad3 when the cardiac underwent PD-1 inhibitor in conjunction with thoracic irradiation (IR). The findings of this research indicate that PD-1 inhibitor worsen RIMF in mice by triggering GSDMD-induced pyroptosis and influencing the TGF-β1/Smads pathway. While using the caspase-1 inhibitor Z-YVAD-FMK, RIMF can be alleviated. Blocking GSDMD may be a viable strategy for managing myocardial fibrosis caused by the combination of PD-1 inhibitors and radiotherapy.

The purpose is to compare the clinical efficacy and toxicity of etoposide plus lobaplatin (EL) or etoposide plus cisplatin (EP) with concurrent thoracic radiotherapy during the treatment of limited-stage small cell lung cancer (LS-SCLC). Forty-two patients with LS-SCLC were randomly divided into EL ( n = 19) or EP ( n = 23) regimens combined with thoracic intensity-modulated radiotherapy. The primary endpoint was 1-year progression-free survival (PFS) rate. The 1-, 2-, and 3-year PFS rates in the EL and EP cohorts were 50.8, 38.1, and 12.7%; and 56.5, 43.5, and 29.0%, respectively ( P = 0.527), whereas the 1-, 2-, and 3-year overall survival (OS) rates were 72.2, 52.5, and 43.8%; and 73.9, 48.4, and 48.4%, respectively ( P = 0.923). The hematological toxicities were similar in two cohorts. However, gastrointestinal reactions were more severe in the EP group. The incidence of nausea and vomiting in EL and EP cohorts were 31.6% vs. 73.9% ( P = 0.006) and 20.1% vs. 60.9% ( P = 0.009), respectively. The two cohorts did not show ≥grade 4 radiation esophagitis and ≥grade 3 radiation pneumonitis. The incidence of acute radiation esophagitis in EL group was lower ( P = 0.038), both groups showed a similar incidence of radiation pneumonitis ( P = 1.000). EL or EP chemotherapy with concurrent thoracic radiotherapy showed similar PFS and OS. The EL group showed milder gastrointestinal toxicity and radiation esophagitis. Radiation pneumonitis and hematological toxicity were similar in the two regimens, which can be tolerated by patients.

目的:通过小鼠动物模型,观察程序性死亡受体1(programmed death 1,PD-1)抑制剂对肺脏免疫微环境、肺损伤及纤维化的影响.方法:将15只C57BL/6小鼠随机分为3组,每组5只,A组为空白对照组,B组为IgG组对照组,C组为PD-1抑制剂组,每周给药1次,共计给药6周.第7周麻醉处死小鼠,HE和Masson染色观察肺组织形态学改变和评估纤维化,免疫组化检测CD3+、CD4+、CD8+T淋巴细胞浸润情况,流式细胞术检测细胞因子(IL-4、IL-6、IL-17A、TNF-α、TGF-β1、IFN-γ)水平,并检测肺组织羟脯氨酸含量.结果:A组和B组未见明显的肺损伤,C组可见肺泡间隔增厚、间质中炎症细胞浸润增加.A组和B组未见明显纤维化,C组可见肺间质胶原纤维沉积,半定量分析结果显示A、B、C组胶原容积分数(collagen volume fraction,CVF)分别为4.30％±1.06％、5.10％±1.37％、10.70％±2.83％,C组的CVF高于A组和B组(P<0.01);A组和B组的羟脯氨酸含量相似,C组羟脯氨酸含量比A组和B两组增高(P<0.01).与A组和B组比较,C组的CD3+T淋巴细胞浸润显著增加,主要是以CD8+T淋巴细胞浸润为主,CD4+T淋巴细胞浸润不明显,C组的IL-6、TGF-β1水平高于A组和B组(P<0.01).结论:PD-1抑制剂促进CD8+T淋巴细胞向肺组织浸润,通过诱导免疫炎症反应导致肺损伤及纤维化.

Purpose:To explore the effect of PD-1 inhibitors combined with irradiation on myocardial injury and the changes of HMGB1-associated inflammatory markers.Methods:Four groups of five mice were used, each groupformed by randomly dividing 20 mice (group A control; group B PD-1 inhibitors; group C Irradiation; group D PD-1 inhibitors+irradiation; n = 5 for each). The mice were treated with either PD-1 inhibitors or a 15 Gy dose of single heart irradiation, or both. Hematoxylin-eosin staining assessed the morphology and pathology of heart tissue; Masson staining assessed heart fibrosis; Tunel staining evaluated heart apoptosis; flow cytometry detected CD3+, CD4+, and CD8+ T lymphocytes in heart tissues; enzyme linked immunosorbent assay evaluated IL-1β, IL-6, and TNF-ɑ of heart tissue; Western blot and quantitative real-time PCR (qPCR) detected the expression of protein and mRNA of HMGB1, TLR-4, and NF-κB p65 respectively.Results:The degree of heart injury, collagen volume fraction (CVF) and apoptotic index (AI) in groups B, C, and D were higher than group A, but the differences between the CVF and AI of group A and group B were not statistical significance (P>0.05). Similarly, the absolute counts and relative percentage of CD3+ and CD8+ T lymphocytes and the concentrations of IL-1β, IL-6, and TNF-α in heart tissue with group D were significantly higher than the other groups (P<0.05). In addition, compared with group A, the expression of protein and mRNA of HMGB1 and NF-κB p65 in other groups were higher, and the differences between each group were statistically significant while TLR4 was not. In addition, interaction by PD-1 inhibitors and irradiation was found in inflammatory indicators, especially in the expression of the HMGB1 and CD8+ T lymphocytes.Conclusion:PD-1 inhibitors can increase the expression of HMGB1-associated inflammatory cytokines and aggravate radiation-induced myocardial injury.

PURPOSE:This study was designed to evaluate the effects of PD-1 inhibitor on lung tissue morphology and the immune system in a mouse model of radiation-induced lung injury (RILI) and to assess interactions between radiation therapy and PD-1 inhibition.METHODS:Twenty C57BL/6 mice were divided randomly into four groups of five mice each. Mice were treated with an anti-mouse PD-1 monoclonal antibody, whole thorax irradiation, both or neither. Lung tissue morphology and pathological changes were assessed by hematoxylin-eosin staining; lung fibrosis was assessed by Masson staining and analysis of hydroxyproline; CD3+, CD4+, and CD8+ T lymphocytes in lung tissues were detected immunohistochemically; and the concentrations of transforming growth factor-β1 (TGF-β1) and interleukin-6 (IL-6) in lung tissue were evaluated by cytokine multiplex analysis.RESULTS:Lung injury scores and indicators of pulmonary fibrosis were higher in mice administration whole thorax irradiation than in control mice. Inflammatory infiltrate scores, alveoli deformation scores, collagen volume fractions and hydroxyproline contents in lung tissues were all significantly higher in mice administered PD-1 inhibitor plus irradiation than in the other three groups. Similarly, the percentages of CD3+ and CD8+T cells and the concentrations of IL-6 and TGF-β1 in lung tissue were significantly higher in mice treated with radiation and PD-1 inhibitor than in the other groups. However, PD-1 inhibitor and irradiation interacted significantly only in the elevation of TGF-β1 level.CONCLUSION:Whole thorax X-ray irradiation in mice can cause pulmonary injury and fibrosis, which could be exacerbated by PD-1 inhibitors. Radiotherapy combined with PD-1 inhibitors may aggravate RILI by synergistically upregulating TGF-β1 expression, thereby affecting the immune-inflammatory microenvironment in the lungs.

目的:探讨复方斑蝥注射液联合放化疗在局部晚期鼻咽癌治疗中的应用价值.方法:以我中心2015年7月至2017年4月收治的157例初治局部晚期鼻咽癌患者为研究对象,将其随机分为试验组79例与对照组78例.试验组患者采用复方斑蝥注射液联合同步放化疗,对照组患者行常规同步放化疗治疗.收集临床资料,比较两组患者治疗期间相关不良反应发生率、机体免疫功能变化和近期疗效情况.结果:试验组与对照组的CR、PR、SD、PD差异无统计学意义(P=0.269>0.05),近期有效率(CR+PR)差异存在统计学意义(P=0.001),试验组较优于对照组;进一步分析两组患者的4级不良反应发生率存在统计学差异(P<0.05),两组患者治疗前后CD3+、CD4+、CD4+/CD8+、CD8+、CD19+淋巴细胞计数下降,试验组较对照组下降趋势缓和.结论:局部晚期鼻咽癌在规范抗肿瘤治疗过程中给予复方斑蝥注射液治疗可降低患者治疗期间严重不良反应的发生率,对机体免疫功能可能有一定的保护作用,并在一定程度上可辅助提高疗效,需开展多中心、大样本临床研究进一步证实.