MDSC介导的免疫抑制在骨髓增生异常综合征中作用的研究

Myelodysplastic syndromes (MDS) is a group of clonal hematopoietic stem cell disorder. The patients with MDS have increased malignant stem cells (leukemia stem cell, LSC) which correlated with MDS stages. And the number and function of many immune cells subsets in MDS are altered. It suggestes immunosuppression is one of important mechanisms on immune evasion of LSC in MDS. Myeloid-derived suppressor cells (MDSC) have been proved to be a key role on tumor immune evasion, which aggravate the development and growth of tumor. In past study, we found that circulating MDSC were increased in MDS patients and correlated with MDS severity. We plan to investigate the role of MDSC on immune suppression in MDS patients and NUP98–HOXD13 mice. The proportion of MDSC in bone marrow of MDS patients and health controls will be measured by flow cytometry (FCM). The karyotypes of MDSC will be measured by fluorescence in situ hybridization (FISH). The STAT3 and PD-L1/PD-1 pathway will be measured by FCM. The ARG1/iNOS activity, standing for the activity of MDSC, will be measured by RT-PCR and western blot. The apoptosis and function of CTL will be measured after co-culture with MDSC. The cytotoxicity of CTL on K562 cell line inhibited by MDSC will be observed in vitro. After interference of ARG1/iNOS (by RNA interference) and PD-L1 (by anti-PD-L1 mAb), the function of MDSC will be detected. The proportion and function of MDSC in NUP98–HOXD13 mice will be measured in different period. After using anti-GR-1 mAb, the development of MDS in NUP98–HOXD13 mice will be compared with the controls. We want to clarify the role of MDSC in pathogenesis of MDS, which will probably to a novel target for treating MDS in future.

骨髓增生异常综合征(MDS)是克隆性造血干细胞疾病，其恶性克隆(LSC)随疾病进展而增加，但LSC免疫逃逸的机制尚不清楚。我们已发表的研究提示，在肿瘤免疫逃逸中占重要地位的髓源性抑制细胞(MDSC)在MDS患者外周血升高，并与疾病恶性程度相关，可能是抑制正常免疫、促进LSC免疫逃逸与疾病进展的重要环节。本研究拟通过MDS患者与小鼠模型，进一步研究MDSC介导的免疫抑制作用。主要目标：①检测MDS患者骨髓原位MDSC的生物学特性，包括数量、克隆来源、信号通路pSTAT3与功能酶ARG1/iNOS；②分析MDSC对CTL数量与功能的抑制作用；③分析MDSC抑制CTL的机制，包括PD-L1/PD-1途径检测、抑制ARG1/iNOS后的功能变化；④检测MDS小鼠模型在疾病不同阶段MDSC的变化，以及去除MDSC对疾病进程的影响。阐明MDSC在MDS中的作用与功能途径，为MDS靶向治疗提供新思路。

骨髓增生异常综合征(MDS)是克隆性造血干细胞疾病，其恶性克隆(LSC)随疾病进展而增加，但LSC免疫逃逸的机制尚不清楚。我们已发表的前期工作提示，在肿瘤免疫逃逸中占重要地位的髓源性抑制细胞(MDSC)在MDS患者外周血升高，并与疾病恶性程度相关，可能是抑制正常免疫、促进LSC免疫逃逸与疾病进展的重要环节。本研究采用流式细胞术（FCM）、荧光免疫原位杂交（FISH）、RT-PCR、Western blot等技术，通过对比MDS患者与正常人的骨髓MDSC（Lin-HLA-DR-CD33+）的数量与功能，以及通过MDS小鼠模型的体内实验，即NUP98–HOXD13小鼠模型（NHD13小鼠），阐明MDSC介导的免疫抑制在MDS发病机制中的作用。结果发现：①MDS患者骨髓原位的MDSC数量增高，与疾病危险度相关，并存在细胞遗传学异常，其STAT3活化增强、功能酶ARG1表达增高、抑制性分子PD-L1表达增高，而MDS患者血清中STAT3通路相关细胞因子IL-6、CCL2水平增高；同时应用地西他滨治疗后的MDS患者MDSC的STAT3活化与PD-L1分子表达均下调，提示其具有表观遗传学改变；②MDS患者细胞毒性T细胞（CTL）表达抑制性分子PD-1增高，同时其活性分子CD3ζ表达降低，提示CTL功能受抑；③通过FCM及免疫磁珠分选MDSC与CTL，进行共培养后发现，MDSC可抑制CTL数量与功能（凋亡增加、分泌细胞因子减少），进一步实验发现，通过抑制STAT3通路、PD-L1/PD-1通路，可下调MDSC对CTL的抑制作用；④动物实验结果显示，与对照组（野生型）相比，NHD13小鼠体内MDSC（GR-1+CD11b+）比例增高，且高表达PD-L1分子，同时CTL、T辅助细胞（Tfh）表面高表达PD-1分子，验证了MDSC通过PD-L1/PD-1通路介导的免疫异常可能为恶性克隆免疫逃逸的重要环节。综上，我们的研究表明，MDS患者MDSC数量和功能均亢进，对CTL具有免疫抑制作用，而STAT3通路、PD-L1/PD-1通路为其上游调控因素，抑制上述通路可抑制MDSC介导的对CTL的免疫抑制作用。本研究进一步阐述了MDSC在MDS中的作用与功能途径，为研发MDS新的靶向治疗提供了实验依据。