抑制Notch1信号在分离aGVHD和GVL中的作用研究

Acute graft-versus-host disease (aGVHD) is a potentially life-threatening complication that is mediated by both host-derived antigen presenting cells (APCs) and donor T cells after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Despite prophylaxis and treatments, aGVHD still occurs in many allo-HSCT patients. Furthermore, standard suppressive therapy for aGVHD impairs graft-versus-leukemia (GVL) activity, increasing the risk of tumor relapse. Notch1 signal is highly concerned recently for it not only controls host-derived APCs differentiation and function, but also regulates differentiation of Th1 and Th2 CD4+ T-cell. Therefore, the role of Notch1 signal inhibition becomes more and more important in aGVHD study. In our previous study, we found that Notch1 inhibition decreased Foxp3 expression in Jurkat cell line, which might induced immune tolerance. This study is to investigate the role of Notch1 inhibition by γ-secretase inhibitor DAPT in aGVHD and GVL as well as in separation of aGVHD from GVL in murine aGVHD model. The degree of clinical and histopathologic GVHD is assessed and the functions of host-derived APCs and donor T cells are analyzed in vitro and in vivo by flow cytometry, ELISPOT, ELISA and Q-PCR. This study will open a way to aGVHD prophylaxis and treatments while preserving GVL in the future.

急性移植物抗宿主病（aGVHD）是allo-HSCT后常见的具有潜在致死性的并发症，由宿主抗原递呈细胞（APCs）和供者T细胞共同介导对受者组织器官造成免疫损伤。aGVHD的预防和治疗削弱了移植物抗白血病（GVL）效应，增加了肿瘤复发率。Notch1信号不仅能调节宿主APCs的分化和功能，还能指导T细胞向Th1或Th2分化，成为防治aGVHD的新亮点。本课题组前期研究发现抑制Notch1信号能下调T细胞淋巴瘤/白血病细胞中Foxp3的表达，提示Notch1可能参与肿瘤的免疫耐受。本课题以前期结果：Notch1信号参与T细胞调节为基础，通过建立抑制Notch1信号的小鼠aGVHD和GVL模型，通过流式细胞技术、酶联免疫斑点法、酶联免疫吸附试验、活体成像和PCR等了解抑制Notch1信号在aGVHD和GVL中的作用，为防治aGVHD、保持GVL效应提供新方法。

项目背景：急性移植物抗宿主病（aGVHD）是allo-HSCT（异基因造血干细胞移植）后常见的具有潜在致死性的并发症，由宿主抗原递呈细胞（APCs）和供者T细胞共同介导对受者组织器官造成免疫损伤。aGVHD的预防和治疗削弱了移植物抗白血病（GVL）效应，增加了肿瘤复发率。Notch1信号不仅能调节宿主APCs的分化和功能，还能指导T细胞向Th1或Th2分化，成为防治aGVHD的新亮点。研究内容：拟建立抑制Notch1信号的小鼠aGVHD和GVL模型，通过流式细胞技术、酶联免疫吸附试验和PCR等了解抑制Notch1信号在aGVHD和GVL中的作用，为防治aGVHD、保持GVL效应提供新方法。重要结果及关键数据：1、本课题成功建立小鼠aGVHD模型。20例小鼠allo-HSCT后存活时间均大于20天，aGVHD发病率为100%，发病中位时间为12天。DAPT处理小鼠和对照组小鼠aGVHD临床总评分为4分。体内DAPT处理小鼠体重下降百分比较对照组小鼠明显减少（p<0.05）；体内、外DAPT处理组小鼠间体重下降无显著差异。小鼠aGVHD模型中，受体鼠肝、皮肤和肠黏膜均可见较多淋巴细胞浸润。DAPT处理小鼠外周血Hes1 mRNA表达均较对照组小鼠明显下降，提示Notch信号受抑。Notch信号受抑小鼠骨髓和脾脏单个核细胞CD11c、CD11b、CD86、PDL1/CD274、CD40和CD8比例较对照组下降，其中以体外实验组小鼠下降明显，提示体外抑制Notch信号可抑制与抗原提呈相关的树突状细胞功能。其次，CD3、CD4、CD25、CD44和Foxp3比例较对照组明显升高，以体内实验组小鼠升高为主，提示体内抑制Notch信号可能诱导T细胞向Th2或调节性T细胞分化，从而减轻aGVHD反应。血清IFN-γ细胞因子水平与对照组小鼠比较无显著差异。2、移植后向受体BALB/c小鼠腹腔注射A20细胞，成功建立C57BL/6（B6, H-2b）→BALB/c（H-2d）小鼠GVL模型。11例小鼠均成瘤，成瘤中位时间为13天（11-19天）。科学意义：1）DAPT能抑制Notch1信号通路；2）抑制Notch1信号通路可一定程度减轻小鼠aGVHD，其机制可能与下调抗原提呈细胞功能和诱导T细胞向Th2或调节性T细胞分化相关；3）其GVL保护效应正继续探讨中。