抗供者特异性抗体(DSA)对造血干细胞移植预后的影响及其机制研究

Recent study confirmed that the donor- specific antibody(DSA) was associated with the prognosis after HSCT, but the mechanism was unclear. Our study found that the presence of DSA pre-transplant and de dove of DSA post-transplant(include of anti-HLA and anti-MICA specific antibodies) were the most important molecular impact on outcomes of imbedded, retard of hematopoietic reconstitution,chronic rejection, complication of transplant and failure of transplant in HSCT. So we speculated that high titer DSA could activate PI3K-Akt-mTOR1 and ERK cell signaling pathway, regulate endothelial cells proliferation. The p70S6K and ERK phosphorylation were the most important factors which lead to functional impaired of ECs. This project is going to investigate recipients of high, middel and low DSA expression and perform HLA and MICA gene typing for both recipients and donors. The result will illustrate that high titer DSA might be the most important fundamental impact on outcomes of HSCT. Also this project is going to establish the DSA experimental model and animal model in vitro and in vivo to explore the mechanism of DSA development in combination with clinical data analysis using sequence-based testing,Luminex, flow cytometry, Western blot and real-time PCR methods. It is demonstrated that high titer DSA play a crucial role in prognosis of HSCT providing experimental foundation for new protocol for clearance of DSA by combination with Bortezomib and CD20 Mcabs.These findings provide new idea and significance that may be beneficial to improve the clinical outcomes of HSCT and prevention DSA.

临床研究表明抗供者特异性抗体(donor specific anti-antibody，DSA)与HSCT的预后相关，但其机制尚不清楚。申报者的前期研究发现：受者预存和新生DSA（抗HLA和MICA抗体）是影响HSCT植入、造血重建、慢性排异、移植并发症的重要分子。据此推测：高滴度DSA激活ERK和PI3K-Akt-mTOR1通路调控内皮细胞的增殖，p70S6K和ERK磷酸化是导致受者内皮细胞功能受损的主要因素。本项目将从受者高、中、低表达DSA和供受者HLA和MICA基因错配的角度，拟建立体外表达DSA的实验模型和应用重组蛋白建立新生DSA的动物模型，并结合临床病例，探讨DSA发生、发展的机制，阐明高滴度DSA是影响HSCT预后的根本,为采用硼替佐米和CD20单抗联合新方案清除DSA提供实验依据。本项目的临床意义将为判断HSCT预后和个体化治疗提供新的重要指标，为防治DSA提供新思路。

HLA抗体是影响同种异体移植预后的重要因素，因此如何有效清除抗 HLA 抗体及抑制抗体介导的排斥反应已经成为研究和临床治疗的热点。本课题围绕体内实验、体外实验、动物模型完成如下研究成果：⑴体内实验研究证实移植前预存抗HLA抗体阳性患者，尤其是在HLA-I类抗体阳性，及HLA-A和Cw抗体阳性患者中，移植后巨核系造血重建延缓、II-IV度急性和慢性GVHD发生率增加、移植后死亡率升高、总生存率下降；尤其是在AML/MDS患者中，移植前后持续抗HLA抗体阳性患者，其移植预后明显不良。因此，该研究结果提示移植前预存抗HLA抗体是影响移植预后的最危险因素之一，故在临床需要在移植前常规检测HLA抗体及抗供者特异性抗体(DSA)分析。⑵首次在国内报道成功建立DSA与内皮细胞共培养体外实验细胞模型，研究表明不同强度和不同浓度的DSA均可激活内皮细胞PI3K/AKT信号通路，采用Westren Blot和流式细胞术等方法检测PI3K/AKT信号通路增殖和抗凋亡蛋白的表达及细胞的增殖率，证实DSA引起内皮细胞增殖、存活和凋亡等改变，为证实体内研究结论提供了理论依据。同时发现在HLA-I类位点中HLA-A、C位点的抗原性可能强于HLA-B位点，由于HLA抗原强弱性存在差异，尤其因接受供受者HLA基因错配移植患者，新生DSA易与受者体内表达供者HLA抗原的组织和细胞发生免疫反应，成为影响移植后排异、移植相关并发症和导致植入失败的重要因素，此研究成果为在供受者HLA基因错配移植选择最佳错配供者提供了实验依据。⑶通过构建预存HLA-A2抗体阳性BAL b/c小鼠模型，使用硼替佐咪和利妥昔单抗等药物治疗致敏小鼠，检测小鼠外周血HLA-A2抗体、小鼠脾脏、骨髓和外周血中的淋巴细胞数、CD138+ 浆细胞及C19+B 细胞亚群变化，研究结果表明硼替佐咪长期治疗可以有效降低 HLA-A2 抗体滴度、骨髓中前/祖 B 细胞和脾脏中淋巴细胞，及CD138+ 浆细胞。本研究项目已从分子、细胞、组织、动物整体水平并结合临床资料多方面，研究抗HLA抗体和DSA在HSCT中影响移植预后不良的致病机制，并探索清除抗体新的干预治疗措施，为临床改善患者移植预后提供了新的策略和思路。