移植物化学修饰与阻断 OX40-OX40L途径预防异基因骨髓移植小鼠移植物抗宿主病的研究

目的 观察甲氧基聚乙二醇-琥珀酰亚胺丙酸酸酯(mPEG-SPA)与抗OX40L单抗预处理移植物对小鼠异基因骨髓移植后急性移植物抗宿主病(aGVHD)的影响及其机制.方法 供、受鼠的脾淋巴细胞体外混合培养,加入抗OX40L单抗孵育培养后,再用mPEG-SPA化学修饰,与C57BL/6供鼠骨髓细胞混合后移植给经致死量全身照射的BALB/c受鼠.同时设相应的对照组.观察移植后受鼠aGVHD的临床表现、病理学改变、存活时间及生存率等,检测移植鼠T淋巴细胞亚群,细胞因子的变化及异基因嵌合体.结果 ①单纯骨髓移植对照组小鼠均出现aGVHD临床表现,17 d内全部死于aGVHD,存活时间为(12.1±5.5)d;而mPEG-SPA和抗OX40L单抗单独或联合预处理组小鼠一般状态较好,部分出现aGVHD表现,且较单纯骨髓移植对照组症状轻,皮肤、肝脏、小肠病理表现均较单纯骨髓移植对照组明显减轻,mPEG-SPA和抗OX40L单抗单独或联合预处理组小鼠存活时间分别为(36.2±24.9)、(32.0±24.8)和(44.3±23.2)d,均较单纯骨髓移植对照组明显延长(P＜0.05),其中抗OX40L mPEG-SPA联合预处理组小鼠平均存活时间最长(P＜0.05),mPEG-SPA单独预处理组和抗OX40L单独预处理组牛存率分别为50.0%、41.7%,明显高于单纯骨髓移植组,OX40L mPEGSPA联合预处理组生存率最高,为66.7%.②移植后对照组小鼠血清IFN-γ浓度升高,在+10～+15d时达高峰;而mPEG-SPA和抗OX40L单抗单独或联合预处理组降低,+10 d时降至最低,OX40LmPEG-SPA联合预处理组降低最明显(P＜0.01).移植后对照组IL4、IL-10浓度轻度降低,而mPEG-SPA和抗OX40L单抗单独或联合预处理组均明显升高(P＜0.05),+10～+15 d达高峰,以联合组升高最明显(P＜0.01).③mPEG-SPA和抗OX40L单抗单独或联合预处理组+60 d时异基因嵌合率为95%～100%,证实为完全供者型植入.结论 mPEG-SPA与抗OX40L单抗联合应用能够阻断T细胞激活的抗原和共刺激双信号通路,协同抑制T细胞的增殖活性,促进Th0细胞向Th2细胞分化,从而明显减轻小鼠骨髓移植中aGVHD的发生.

Prophylaxis of graft-versus-host disease in mice by chemical modification of graft and OX40-OX40L costimulatory pathway

Objective To explore the prophylaxis effect of pretreatment of allograft with methoxypoly-ethylene glycol-suceinimidyl-propionic acid ester (mPEG-SPA) and anti-OX40L monoclonal antibody (McAb) on acute graft-versus-host disease (aGVHD)after allogeneic bone marrow transplantation (allo-BMT) in mice.Methods Responder splenocytes from C57BL/6 donor mice (H-2~b) were co-cultured with stimulator splenocytes from BALB/c recipient mice (H-2~d) for 7 days in the presence or absence of anti-OX40L McAb followed by mPEG-SPA chemical modification.Donor bone marrow cells plus the mixed culture of T-cells were then transplanted into lethally irradiated BALB/c mice.The BALB/c recipient mice were divided into four groups:group A(allo-BMT control group),group B (mPEG-SPA modification group).group C (anti-OX40L McAb pretreated group) and group D (mPEG-SPA and anti-OX40L McAb dual-treated group).Survival time and survival rate of the recipients were observed after allo-BMT.GVHD was assessed by clinical signs and histological changes of skin,liver and small intestines.Enzyme-linked immunosorbent assay (ELISA) was used to detect cytokines (IL-4,IL-10 and INF-γ) production.Flow cytometry (FCM) analysis was used to detect allogeneic chimerism.Results ①The mice in group A developed typical clinical signs of aGVHD and all mice died within 17 days after BMT with an average survival time(AST) of (12.1±5.5) days.The signs of aGVHD were less evident in mice of groups B,C and D,and their AST (36.2 ±24.9,32.0±24.8 and 44.3±23.2 days,respectively) were all longer than that in group A (P <0.05).AST of group D being the longest (P <0.05).The survival rates at day 60 post-BMT in groups B,C and D were 50%,41.7% and 66.7%,respectively. ②Serum IFN-γ level was increased after BMT in group A,and peaked in day 10 to day 15 post-BMT,while the level was decreased in groups B,C and D,reached the nadir on the day 10 post-BMT,with the lowest in group D (P<0.01).After BMT,IL-4 and IL-10 levels were slightly decreased in group A,their levels were elevated in groups B and C (P <0.05) and even more significantly increased in group D (P<0.01).IL-4 and IL-10 levels peaked between day 10 and 15 post-BMT.③ The average proportion of H-2~b positive cells in recipient mice was 95%-100% on day 60 post-BMT,with complete donor-type implantation.Conclusion Combination of mPEG-SPA and anti-OX40L McAb can block T-cell activated antigens and co-stimulatory pathway,regulate the T cells differentiation and induce the immune shift of Th0 cells toward Th2 cells.The immune tolerance induced by this method can significantly relieve aGVHD after allo-BMT.