PD-1单克隆抗体加重allo-HSCT小鼠炎性损伤和cGVHD的机制研究

Chronic graft-versus-host disease in inflammatory mice and mechanism of PD-1 monoclonal anti-body exacerbating the disease

目的探讨程序性死亡受体1单克隆抗体(PD-1单抗)加重allo-HSCT小鼠炎性损伤和慢性移植物抗宿主病(chronic graft versus host disease,cGVHD)的作用机制。方法将DBA/2小鼠的骨髓和脾细胞注射到白消安联合氟达拉滨化疗方案预处理BALB/C小鼠构建cGVHD模型(n=48),将BALB/c(H-2Kd)小鼠按随机数字法分为炎性组(n=8)和对照组[注射磷酸盐缓冲液(PBS),n=8],使用酵母多糖诱导炎性组小鼠为炎症状态。观察记录两组小鼠移植后体重变化、生存情况以及cGVHD的表现,另取两组小鼠提取受累靶器官(皮肤、肝脏、小肠、结肠、肺脏)组织进行病理评分(每组n=4);体外实验取8~12周的BALB/C小鼠按上述方法建立炎症cGVHD模型,同样按照随机数字法分成PD-1组(注射PD-1单克隆抗体)和对照组(每组n=12),观察记录两组小鼠移植后体重变化、生存情况以及cGVHD的表现,另取小鼠应用流式细胞技术检测靶器官的细胞亚群、表面的共刺激分子等(n=4)。结果炎性组呈现更显著的cGVHD改变,小鼠死亡率高,体重下降快,GVHD的症状更严重。炎性组小鼠肝脏和肺脏的替代活化的巨噬细胞(M2)比例明显低于对照组(P<0.05),经典活化的巨噬细胞(M1)无差别。在体外实验中,炎性组相较对照组脾细胞M1比例均增多,M2比例明显均减少。分泌的白细胞介素(IL)-4,IL-10减少,共刺激分子CD80和CD86增多。结论PD-1单抗可加重allo-HSCT小鼠的炎性损伤和cGVHD,其作用机制可能与调节性T细胞(regulatory cell,Treg)减少相关。

Objective To explore the mechanism of exacerbating chronic graft versus host disease(GVHD)in mice with inflammatory status and enhancing immune injury in mice with PD-1.Method Bone marrow and spleen cells of DBA/2 mice were injected into BALB/C mice pretreated with chemotherapy regimen(Flu+Bu)for constructing a chronic GVHD model.The animals were assigned into two groups of zymosan(100M SPL+10M BM+Zymosan)and control(100M SPL+10M BM+PBS).After transplantation,two groups of mice were observed for weight changes,survival status and chronic GVHD manifestations.Target organ tissues were harvested for pathological scoring.Flow cytometry was employed for detecting cell subpopulations and surface co-stimulatory molecules in target organs.PD-1 monoclonal antibody was injected into inflammatory murine model.Mice were observed and target organ cells were harvested for subsets and co-stimulatory factors.Result In in vivo experiments,zymosan group showed more significant changes of chronic GVHD with higher mortality rate,faster weight loss and more severe symptoms of GVHD.At Week 2 post-transplantation,hematoxylin-eosin stain of target organ tissue was performed for pathology examination.Zymosan group showed more lymphocyte infiltration,more severe inflammation and more significant tissue injury with higher GVHD pathological score.The proportion of M2 in liver/lung of zymosan group was significantly lower than that of control group(P<0.05)and no significant difference existed in the proportion of M1.In in vivo experiments,M1 ratio of splenic cell spiked markedly in zymosan group as compared to control group while M2 ratio declined greatly.The secretions of IL-4 and IL-10 dropped significantly while co-stimulatory molecules CD80 and CD86 rose obviously.Conclusion The worsening graft-versus-host disease in inflammatory mice with anti-PD1 treatment is associated with a decline of Treg proportion.